Zika vaccines and immunogenic compositions, and methods of using the same

ABSTRACT

The present disclosure relates to Zika virus vaccines and immunogenic compositions having one or more antigens from a Zika virus (e.g., a Zika virus clonal isolate, a non-human cell adapted Zika virus, etc.), and methods of treatments and uses thereof

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under Contract No.HHSO100201600015C with the Department of Health and Human Services,Office of the Assistant Secretary for Preparedness and Response,Biomedical Advanced Research and Development Authority. This inventionwas created in the performance of a Cooperative Research and DevelopmentAgreement with the Centers for Disease Control and Prevention, an Agencyof the Department of Health and Human Services. The Government of theUnited States has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates to Zika virus vaccines and immunogeniccompositions having one or more antigens from a Zika virus (e.g., a Zikavirus clonal isolate, a non-human cell adapted Zika virus, etc.) andmethods of treatment and uses thereof.

BACKGROUND

Zika virus, a flavivirus classified with other mosquito-borne viruses(e.g., yellow fever, dengue, West Nile, and Japanese encephalitisviruses) within the Flaviviridae family has spread rapidly in ahemispheric-wide epidemic since the virus was introduced into Brazil in2013. The virus has reached the Central and North Americas, includingterritories of the United States, consequently now threatening thecontinental US. Indeed, Zika virus strain PRVABC59 was isolated fromserum from a person who had traveled to Puerto Rico in 2015. The genomeof this strain has been sequenced at least three times (See Lanciotti etal. Emerg. Infect. Dis. 2016 May; 22(5):933-5 and GenBank AccessionNumber KU501215.1; GenBank Accession Number KX087101.3; and Yun et al.Genome Announc. 2016 Aug. 18; 4(4) and GenBank Accession NumberANK57897.1).

Initially isolated in 1947 in Uganda, the virus was first linked tohuman disease in 1952, and has been recognized sporadically as a causeof mild, self-limited febrile illness in Africa and Southeast Asia(Weaver et al. (2016) Antiviral Res. 130:69-80; Faria et al. (2016)Science. 352(6283):345-349). However, in 2007, an outbreak appeared inthe North Pacific island of Yap, and then disseminated from island toisland across the Pacific, leading to an extensive outbreak in 2013-2014in French Polynesia, spreading then to New Caledonia, the Cook Islands,and ultimately, to Easter Island. An Asian lineage virus wassubsequently transferred to the Western Hemisphere by routes that remainundetermined (Faria et al. (2016) Science. 352(6283):345-349). The virusmay be transmitted zoonotically by Aedes aegypti, A. albopictus, andpossibly by A. hensilli and A. polynieseinsis (Weaver et al. (2016)Antiviral Res. 130:69-80). Additionally, it is thought that othervectors for transmitting the virus may exist, and the virus may betransmitted by blood transfusion, transplacentally, and/or throughsexual transmission.

In late 2015, a significant increase in fetal abnormalities (e.g.,microcephaly) and Guillain-Barre syndrome (GB S) in areas of widespreadZika virus infection raised alarm that Zika virus might be much morevirulent than originally thought, prompting the World HealthOrganization (WHO) to declare a Public Health Emergency of InternationalConcern (PHEIC) (Heymann et al. (2016) Lancet 387(10020): 719-21).Although the WHO has since declared an end to the PHEIC, Zika continuesto pose in particular a significant threat for pregnant women and theirunborn babies.

While Zika virus poses a substantial public health threat, noFDA-approved vaccine or treatment currently exists, and the onlypreventative measures for controlling Zika virus involve managingmosquito populations.

In recent efforts to characterize a recombinant Zika virus for thedevelopment of a potential vaccine, a non-human cell adapted Zika viruswas identified that harbors a mutation in the viral Envelope protein atposition 330 (Weger-Lucarelli et al. (2017) Journal of Virology 91(1):1-10). The authors of this study found that full-length infectious cDNAclones of Zika virus strain PRVABC59 were genetically unstable whenamplified during cloning, and opted to split the viral genome to addressthe observed instability, developing and applying a two plasmid system.However, a two plasmid system for the development of a Zika vaccine isless desirable.

BRIEF SUMMARY

Thus, there is a need to develop vaccines and immunogenic compositionsfor treating and/or preventing Zika virus. Accordingly, certain aspectsof the present disclosure relate a vaccine or immunogenic compositioncomprising a dose of 1 μg to 40 μg of (one) antigen from a Zika virus,wherein the antigen is an inactivated whole virus.

Accordingly, certain aspects of the present disclosure relate a vaccineor immunogenic composition comprising a dose of 1 μg to 40 μg of (one)antigen from a Zika virus, wherein the Zika virus comprises at least onenon-human cell adaptation mutation.

Accordingly, certain aspects of the present disclosure relate a vaccineor immunogenic composition comprising a dose of 1 μg to 40 μg of (one)antigen from a Zika virus, the Zika virus having a mutation at position98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQID NO: 1.

To meet the above and other needs, the present disclosure is directed,at least in part, to a genetically stable non-human cell adapted Zikavirus harboring an adaptation in the Non-structural protein 1 (with awild-type Envelope protein), allowing for the use of a singlevirus/viral genome system for vaccine production. The present disclosureis also directed, at least in part, to a Zika virus clonal isolate thatis genetically homogenous and/or has been purified away from one or moreadventitious agents. Accordingly, the present disclosure providesvaccines and immunogenic compositions useful for treating and/orpreventing Zika virus infection (in humans) that include one or moreZika virus antigens (e.g., one or more antigens from a whole inactivatedZika virus) from a Zika virus harboring at least one non-human celladaptation mutation (e.g., a mutation in Zika virus Non-structuralprotein 1) and/or a Zika virus clonal isolate.

The present disclosure is based, at least in part, on the surprisingfinding that both high and low dose vaccines comprising one or moreantigens from separately derived clonal virus populations of non-humancell adapted Zika virus were able to induce robust immune responses andprovide significant protection from Zika virus infection (See Examples 2and Example 6 below). Clonal isolation of the Zika virus strains alsoallowed for: 1) the successful purification of the virus away fromcontaminating agents (e.g., adventitious agents that may be co-purifiedwith the parental strain), and 2) the production of a geneticallyhomogeneous viral population. Moreover, the present disclosure is based,at least in part, on the finding that clonal isolated Zika virusesharboring an adaptation mutation in protein NS1 grew well andpredictably in Vero cells to high titer, and surprisingly, weregenetically stable/genetically homogenous without any detectablemutations in the viral envelope protein (See Examples 1 and 2 below).While a similar mutation in Zika virus Non-structural protein 1 may havebeen observed in the genomic sequencing analysis of 1 out of 3 publishedsequences of Zika virus strain PRVABC59 (Yun et al. Genome Announc. 2016Aug. 18; 4(4)), this reference fails to teach or suggest that a mutationin NS1 may improve stability of the virus; fails to teach or suggestthat a virus harboring the mutation may be used in the development of aneffective vaccine against Zika virus; and fails to teach or suggest thatsuch a vaccine may be effective in inducing a robust immune response andproviding significant protection from Zika virus infection when used atboth low and high doses. Thus, without wishing to be bound by theory,the inventors of the present disclosure have determined that theadaptation mutation in protein NS1 appeared to enhance genetic stabilitywithin the Zika virus, resulting in increased/enhanced replicationefficiency. Further, the Zika strain harboring an adaptation mutation inprotein NS1 was able to be passaged multiple times without developingfurther mutations. Such a stable Zika virus strain is advantageous as amaster virus seed (MVS), or subsequent seeds derived from the MVS, forvaccine production and manufacturing, as the risk of the master virusseed developing undesirable mutations is reduced. Moreover, withoutwishing to be bound by theory, the adaptation mutation in protein NS1 ofthe Zika strain of the present disclosure may also reduce or otherwiseinhibit the occurrence of undesirable mutations, such as a mutationwithin the envelope protein E (Env) of the Zika virus strain.

Accordingly, certain aspects of the present disclosure relate to avaccine or immunogenic composition containing one or more antigen from aZika virus, where the Zika virus contains at least one non-human celladaptation mutation. In some embodiments, the at least one non-humancell adaptation mutation is in Zika virus Non-structural protein 1(NS1). In some embodiments, the at least one adaptation mutation occursat position 98 of SEQ ID NO: 1, or at a position corresponding toposition 98 of SEQ ID NO: 1. In some embodiments, the at least oneadaptation mutation is a Trp98Gly mutation.

Accordingly, certain aspects of the present disclosure relate to avaccine or immunogenic composition comprising a dose of 1 μg to 40 μg of(one) antigen from a Zika virus, wherein the Zika virus comprises atleast one non-human cell adaptation mutation.

Accordingly, certain aspects of the present disclosure relate a vaccineor immunogenic composition comprising a dose of 1 μg to 40 μg of (one)antigen from a Zika virus, the Zika virus having a mutation at position98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQID NO: 1.

In some embodiments that may be combined with any of the precedingembodiments, the at least one adaptation mutation enhances geneticstability as compared to a Zika virus lacking the at least oneadaptation mutation. In some embodiments that may be combined with anyof the preceding embodiments, the at least one adaptation mutationenhances viral replication as compared to a Zika virus lacking the atleast one adaptation mutation. In some embodiments that may be combinedwith any of the preceding embodiments, the Zika virus does not comprisea mutation in Envelope protein E (Env).

In some embodiments that may be combined with any of the precedingembodiments, the non-human cell is a mammalian cell. In some embodimentsthat may be combined with any of the preceding embodiments, thenon-human cell is a monkey cell. In some embodiments, the monkey cell isfrom a Vero cell line. In some embodiments, the Vero cell line is a WHOVero 10-87 cell line.

In some embodiments that may be combined with any of the precedingembodiments, the Zika virus is an African lineage virus or an Asianlineage virus. In some embodiments, the Zika virus is an Asian lineagevirus. In some embodiments, the Zika virus is from strain PRVABC59.

In some embodiments that may be combined with any of the precedingembodiments, the vaccine or immunogenic composition is a purifiedantigen vaccine or immunogenic composition, a subunit vaccine orimmunogenic composition, an inactivated whole virus vaccine orimmunogenic composition, or an attenuated virus vaccine or immunogeniccomposition. In some embodiments, the vaccine or immunogenic compositionis an inactivated whole virus vaccine or immunogenic composition. Insome embodiments, the vaccine or immunogenic composition comprises apurified inactivated whole Zika virus. In some embodiments, the vaccineor immunogenic composition comprises a purified inactivated whole Zikavirus comprising a mutation at position 98 of SEQ ID NO: 1, or at aposition corresponding to position 98 of SEQ ID NO: 1. In someembodiments, the vaccine or immunogenic composition comprises a purifiedinactivated whole Zika virus comprising a Trp98Gly mutation at position98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQID NO: 1. In some embodiments, the vaccine or immunogenic compositioncomprises a purified inactivated whole Zika virus comprising a Trp98Glymutation at position 98 of SEQ ID NO: 1, or at a position correspondingto position 98 of SEQ ID NO:1, wherein the Zika virus is derived fromstrain PRVABC59. In some embodiments, the vaccine or immunogeniccomposition comprises a purified inactivated whole Zika virus comprisinga Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at a positioncorresponding to position 98 of SEQ ID NO:1, wherein the Zika virus isderived from strain PRVABC59 comprising the genomic sequence accordingto SEQ ID NO:2. In some embodiments, the vaccine or immunogeniccomposition comprises a purified inactivated whole Zika which differsfrom strain PRVABC59 in a Trp98Gly mutation at position 98 of SEQ ID NO:1.

In some embodiments that may be combined with any of the precedingembodiments, the virus was chemically inactivated. In some embodiments,the virus was chemically inactivated with one or more of a detergent,formalin, beta-propiolactone (BPL), binary ethylamine (BEI), acetylethyleneimine, methylene blue, and psoralen. In some embodiments, thevirus was chemically inactivated with formalin.

In some embodiments that may be combined with any of the precedingembodiments, the vaccine or immunogenic composition further contains anadjuvant. In some embodiments, the adjuvant is selected from aluminumsalts, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MLA),synthetic lipid A, lipid A mimetics or analogs, MLA derivatives,cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos,lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes,emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG)microparticles, poloxamer particles, microparticles, liposomes, CompleteFreund's Adjuvant (CFA), and/or Incomplete Freund's Adjuvant (IFA). Insome embodiments, the adjuvant is an aluminum salt. In some embodiments,the adjuvant is selected from the group consisting of alum, aluminumphosphate, aluminum hydroxide, potassium aluminum sulfate, andAlhydrogel 85. In some embodiments, at least 75%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% of the one or more antigens are adsorbed to the adjuvant.

In some embodiments that may be combined with any of the precedingembodiments, the vaccine or immunogenic composition is a low or mediumdose vaccine or immunogenic composition (e.g., containing from about 1μg to about 5 μg antigen or 2 μg antigen or 5 μg antigen). In someembodiments that may be combined with any of the preceding embodiments,the vaccine or immunogenic composition is a high dose vaccine orimmunogenic composition (e.g., containing about 10 μg antigen). In someembodiments that may be combined with any of the preceding embodiments,the vaccine or immunogenic composition contains from about 1 μg to about25 μg of the one or more antigens, in particular 2 μg, 5 μg or 10 μg, orin particular 10 μg of the one or more antigens. In certain suchembodiments the antigen is a purified inactivated whole virus, such as aZika virus with a mutation which is a tryptophan to glycine substitutionat position 98 of SEQ ID NO:1 or at a position corresponding to position98 of SEQ ID NO: 1 as described herein. In some embodiments, the vaccineor immunogenic composition comprises a purified inactivated whole Zikavirus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, orat a position corresponding to position 98 of SEQ ID NO:1, wherein theZika virus is derived from strain PRVABC59. In some embodiments, thevaccine or immunogenic composition comprises a purified inactivatedwhole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ IDNO: 1, or at a position corresponding to position 98 of SEQ ID NO:1,wherein the Zika virus is derived from strain PRVABC59 comprising thegenomic sequence according to SEQ ID NO:2. In certain such embodimentsthe Zika virus is a plaque purified clonal Zika virus isolate. In someembodiments that may be combined with any of the preceding embodiments,the vaccine or immunogenic composition contains from about 0.1 μg toabout 100 μg Zika virus antigen or Env. In some embodiments, the vaccineor immunogenic composition is unadjuvanted. In some embodiments that maybe combined with any of the preceding embodiments, the Zika virus is aclonal isolate. In some embodiments, the clonal isolate is substantiallyfree of one or more adventitious agents (e.g., free of one or moreadventitious agents that may be co-purified with the parental strain).

Other aspects of the present disclosure relate to a vaccine comprising aZika virus having a mutation at position 98 of SEQ ID NO: 1, or at aposition corresponding to position 98 of SEQ ID NO: 1. In someembodiments, the vaccine comprises a purified inactivated whole Zikavirus comprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, orat a position corresponding to position 98 of SEQ ID NO:1, wherein theZika virus is derived from strain PRVABC59. In some embodiments, thevaccine comprises a purified inactivated whole Zika virus comprising aTrp98Gly mutation at position 98 of SEQ ID NO: 1, or at a positioncorresponding to position 98 of SEQ ID NO:1, wherein the Zika virus isderived from strain PRVABC59 comprising the genomic sequence accordingto SEQ ID NO:2. In certain such embodiments the Zika virus is a plaquepurified clonal Zika virus isolate.

Other aspects of the present disclosure relate to a vaccine orimmunogenic composition containing: a) an aluminum salt adjuvant; and b)a purified inactivated whole Zika virus, where the Zika virus contains anon-human cell adaptation mutation, and where the non-human celladaptation mutation is a Trp98Gly mutation at position 98 of SEQ ID NO:1, or at a position corresponding to position 98 of SEQ ID NO: 1.

Other aspects of the present disclosure relate to a method of treatingor preventing, in particular preventing Zika virus infection in a humansubject in need thereof, including administering to the human subject atherapeutically effective amount of any of the vaccines or immunogeniccompositions described herein.

Other aspects of the present disclosure relate to a method for inducingan immune response in a human subject in need thereof, includingadministering to the human subject an immunogenic amount of any of thevaccines or immunogenic compositions described herein.

In one aspect the present disclosure relates to a method of treating orpreventing, in particular preventing Zika virus infection in a humansubject in need thereof, comprising administering to the human subjectthe vaccine or immunogenic composition.

In one aspect the present disclosure relates to a method for inducing animmune response against a Zika virus antigen in a human subject in needthereof, comprising administering to the human subject the vaccine orimmunogenic composition.

In one aspect the present disclosure relates to a method of preventingZika virus disease in a human subject in need thereof, comprisingadministering to the human subject the vaccine or immunogeniccomposition. In this case the disease relates to mild fever,maculopapular rash, conjunctivitis and arthralgia. Furthermore, the Zikavirus is a neurotropic flavivirus that can potentially cause diseasewithin the central nervous system and Guillain-Barre Syndrome (GBS).

In one aspect the present disclosure relates to a method of preventingZika virus disease in a fetus or newborn in need thereof, comprisingadministering to the pregnant human subject or a human subject thatintends to become pregnant or woman of childbearing potential thevaccine or immunogenic composition. The Zika disease in this caserelates to serious outcomes for the fetus and newborn. The spectrum ofcongenital anomalies associated with Zika virus infection, known asCongenital Zika Syndrome (CZS), consists of severe microcephaly withpartially collapsed skull, cerebral cortices with subcorticalcalcifications, macular scarring and focal pigmentary retinal mottling,congenital contractures, and marked early hypertonia with symptoms ofextrapyramidal involvement.

In one aspect the present disclosure relates to a vaccine or immunogeniccomposition of for use in a method of treating or preventing, inparticular preventing Zika virus infection in a human subject in needthereof, in a method for inducing an immune response against a Zikavirus antigen in a human subject in need thereof, and in a method ofpreventing Zika virus disease in a human subject, fetus or newborn inneed thereof.

In one aspect the present disclosure relates to the use of the vaccineor immunogenic composition in the manufacture of a medicament for amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject in need thereof, a method for inducing animmune response in a human subject in need thereof, and for a method ofpreventing Zika virus disease in a human subject fetus or newborn inneed thereof.

Within the meaning of this disclosure, the treatment includes thetreatment of a single human subject and the treatment of a human subjectpopulation. A human subject population is considered to encompass thetreatment of more than one individual, e.g. 2 or more individuals.

Accordingly, certain aspects of the present disclosure relates to amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising an antigenfrom a Zika virus, wherein the vaccine or immunogenic composition isadministered as a first (prime) and a second (boost) administrationabout 1 to about 16 weeks apart, and wherein the administration of thevaccine or immunogenic composition induces 14 and/or 28 days after theboost administration geometric mean neutralizing antibody titers in apopulation of at least 20 flavivirus naïve human subjects and/or of atleast 20 Zika virus seronegative human subjects of greater than 300, orgreater than 500, or greater than 1000, or greater than 1500, or greaterthan 2000, or greater than 3000, as determined by the plaque reductionneutralization test (PRNT).

Accordingly, certain aspects of the present disclosure relates to amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising (one) antigenfrom a Zika virus, wherein the vaccine or immunogenic composition isadministered as a first (prime) and a second (boost) administrationabout 1 to about 16 weeks apart, and wherein the administration of thevaccine or immunogenic composition induces 28 days after the boostadministration geometric mean neutralizing antibody titers in apopulation of at least 20 flavivirus naïve human subjects and/or of atleast 20 Zika virus seronegative human subjects, which are at least 10times, or at least 15 times, or at least 20 times, or at least 25 timeshigher than the geometric mean neutralizing antibody titers induced 28days after the prime administration, as determined by the plaquereduction neutralization test (PRNT). The boost administration thusprovides for very high geometric mean neutralizing antibody titersresponsible for a long term protection

Accordingly, certain aspects of the present disclosure relate to amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising (one) antigenfrom a Zika virus, wherein the vaccine or immunogenic composition isadministered as a single dose or prime administration, and wherein theadministration of the vaccine or immunogenic composition induces 14and/or 28 days after the single dose or prime administration geometricmean neutralizing antibody titers in a population of at least 20flavivirus naïve human subjects and/or of at least 20 Zika virusseronegative human subjects of greater than 10, or greater than 50, orgreater than 100, or greater than 200, or greater than 250, asdetermined by the plaque reduction neutralization test (PRNT). The highgeometric mean neutralizing antibody titers indicate an early onset ofprotection, which is beneficial in an outbreak situation or a travelervisiting an endemic area within a short period of time from theadministration of the vaccine or immunogenic composition.

Within the meaning of this disclosure PRNT refers to Zika virusNeutralizing antibody titers in human subjects determined by a plaquereduction neutralization test (PRNT) as described previously (See Sun,W. et al. Protection of Rhesus monkeys against dengue virus challengeafter tetravalent live attenuated dengue virus vaccination. J. Infect.Dis. 193, 1658-1665 (2006). Muthumani K, Griffin B D, Agarwal S, et al.In vivo protection against ZIKV infection and pathogenesis throughpassive antibody transfer and active immunisation with a prMEnv DNAvaccine. NPJ Vaccines 2016; 1: 16021).

Within the meaning of this disclosure the GMT values in human subjectsare in particular to be considered to be measured as the following. Inthe Zika PRNT assay, human serum was 2-fold serially diluted from 1:5 to1:10,240 and mixed with an equal volume of diluted Zika virus (ZIKV)(PRVABC59) to obtain a final dilution of 1:10 to 1:20,480.Neutralization was allowed to proceed 20±2 hours at 2-8° C. after whichthe serum/virus mixture was used to inoculate Vero E6 cells. Virusadsorption was done at 37±2° C. with humidity and CO₂ for 60±10 minutesthen a methylcellulose overlay was added. The infected cells wereincubated at 37±2° C. with humidity and CO₂ for 72±2 hours. Plaques werevisualized by using crystal violet staining and were counted using a CTL(Cellular Technology Limited) reader. Determination of the fifty percentneutralizing titer (PRNT₅₀) was based upon the percent reduction inviral plaques in the presence of serum compared to that of the viruscontrol without serum and was calculated by linear regression. Thetiters represent the reciprocal of the highest dilution resulting in a50% reduction in the number of plaques. Acceptance was assessed byevaluating the virus control (targeting ˜60 pfu/well), cell control,positive control (PRNT₅₀ of 173-658) and negative control (PRNT₅₀<10)tested in parallel with clinical samples. Individual samples andpositive control results were accepted if the correlation coefficient ofthe titration curve generated by linear regression is ≥0.85. Additionalacceptance criteria were based on the quality of the crystal violetstain and plaques generated for the plate or run. PRNT₅₀ results arereported down to the starting dilution of the assay (1:10). PRNT₅₀results that are above the ULOQ will be repeated at a pre-dilution togenerate a result within the quantifiable range of the assay. The resultfrom the pre-diluted sample will be multiplied by the dilution factor togenerate a final result.

Within the meaning of this disclosure seropositivity is defined as titer≥10 as determined by the plaque reduction neutralization test (PRNT);Zika virus seronegative human subjects are subjects with a titer <10 asdetermined by the plaque reduction neutralization test (PRNT),Seroconversion is defined as: Zika virus seronegative human subjects(titer <10) have titer ≥10 post-vaccination as determined by the plaquereduction neutralization test (PRNT); Results <10 as determined by theplaque reduction neutralization test (PRNT) are assigned a titer of 5;Titers between 10 (limit of detection) and 26 (lower limit ofquantification) as determined by the plaque reduction neutralizationtest (PRNT) are assigned a value of 13.

Flavivirus naïve human subjects for the present disclosure are definedto be human subjects without detectable serum antibodies against a panelof flaviviruses, as measured by a reactive antibody based assay(Luminex). Flavivirus screening assay is based on a luminex platform tosimultaneously detect multiple target antigens in the same sample. Thisbead based assay is sensitive, specific and reproducible. For thepresent disclosure, the antigens targeted are Zika, Dengue, Yellowfever, JEV, USUV, SLEV and WNV. Due to cross reactivity amongFlaviviruses, the current antigen set would help detect any priorFlavivirus exposure. References for luminex concept are: Dias D, VanDoren J, Schlottmann S, Kelly S, Puchalski D, Ruiz W, Boerckel P,Kessler J, Antonello J M, Green T, Brown M, Smith J, Chirmule N, Barr E,Jansen K U, Esser M T. 2005. Optimization and validation of amultiplexed Luminex assay to quantify antibodies to neutralizingepitopes on human papillomaviruses 6, 11, 16, and 18. Clin. Diagn. Lab.Immunol. 12:959-969 [PMC free article] [PubMed]. Ayouba A et alDevelopment of a Sensitive and Specific Serological Assay Based onLuminex Technology for Detection of Antibodies to Zaire Ebola Virus. JClin Microbiol. 2017 Dec. 28; 55(1):165-176. doi: 10.1128/JCM.01979-16.

Within the meaning of this disclosure endemic is defined as areas withrisk of infection as defined by the Centers for Disease Control andPrevention, such as for example as of March 2018, namely: Asia:Bangladesh, Burma (Myanmar), Cambodia, India, Indonesia, Laos, Malaysia,Maldives, Pakistan, Philippines, Singapore, Thailand, Timor-Leste (EastTimor), Vietnam. The Pacific Islands: Fiji, Marshall Islands, Papua NewGuinea, Samoa, Solomon Islands, Tonga. The Caribbean: Anguilla; Antiguaand Barbuda; Aruba; Barbados; Bonaire; British Virgin Islands; Cuba;Curacao; Dominica; Dominican Republic; Grenada; Haiti; Jamaica;Montserrat; the Commonwealth of Puerto Rico, a US territory; Saba; SaintKitts and Nevis; Saint Lucia; Saint Martin; Saint Vincent and theGrenadines; Sint Eustatius; Sint Maarten; Trinidad and Tobago; Turks andCaicos Islands; US Virgin Islands. North America: Mexico CentralAmerica: Belize, Costa Rica, El Salvador, Guatemala, Honduras,Nicaragua, Panama South America: Argentina, Bolivia, Brazil, Colombia,Ecuador, French Guiana, Guyana, Paraguay, Peru, Suriname, VenezuelaAfrica: Angola, Benin, Burkina-Faso, Burundi, Cameroon, Cape Verde,Central African Republic, Chad, Congo (Congo-Brazzaville), Côted'Ivoire, Democratic Republic of the Congo (Congo-Kinshasa), EquatorialGuinea, Gabon, Gambia, Ghana, Guinea, Guinea-Bissau, Kenya, Liberia,Mali, Niger, Nigeria, Rwanda, Senegal, Sierra Leone, South Sudan, Sudan,Tanzania, Togo, Uganda. These areas may change.

Accordingly, certain aspects of the present disclosure relate to amethod for inducing an immune response in a human subject population inneed thereof, comprising administering to individual human subjects ofsaid human subject population a vaccine or immunogenic compositioncomprising (one) antigen from a Zika virus, wherein the vaccine orimmunogenic composition is administered as a first (prime) and a second(boost) administration about 1 to about 16 weeks apart, and wherein theadministration of the vaccine or immunogenic composition induces 14and/or 28 days after the boost administration geometric meanneutralizing antibody titers in a human subject population of at least20 flavivirus naïve human subjects and/or of at least 20 Zika virusseronegative human subjects of greater than 300, or greater than 500, orgreater than 1000, or greater than 1500, or greater than 2000, orgreater than 3000, or greater than 5000, or greater than 10,000,determined by the reporter virus particle neutralization assay (RVP).

Accordingly, certain aspects of the present disclosure relate to amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising (one) antigenfrom a Zika virus, wherein the vaccine or immunogenic composition isadministered as a single dose or prime administration, and wherein theadministration of the vaccine or immunogenic composition induces 14and/or 28 days after the single dose or prime administration geometricmean neutralizing antibody titers in a population of at least 20flavivirus naïve human subjects and/or of at least 20 Zika virusseronegative human subjects of greater than 300, or greater than 500,greater than 1000, or greater than 2000, as determined by the reportervirus particle neutralization assay (RVP). The high geometric meanneutralizing antibody titers indicate an early onset of protection,which is beneficial in an outbreak situation or a traveler visiting anendemic area within a short period of time from the administration ofthe vaccine or immunogenic composition.

Within the meaning of this disclosure Reporter virus particle (RVP)neutralization assay refers to Zika Neutralizing antibody titers whichwere analyzed by titration of serum samples with a constant amount ofZika RVPs in Vero cells grown in 96-well plates. RVPs contained the prMEproteins of Zika (strain SPH2012) and a Dengue-based Renilla luciferasereporter. Briefly, sera were heat inactivated at 56° C. for 30 min,diluted, and then incubated at 37° C. with RVPs. The serum/RVP mixturewas then mixed with Vero cells and incubated for 72 hours at 37° C.±2°C./5% CO₂ before detection with luciferase substrate. Data was analyzedusing JMP11 non-linear 4 parameter analysis, normalized to a positivetracking control and effective dose 50% (EC50) was reported.

Accordingly, certain aspects of the present disclosure relate to amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising (one) antigenfrom a Zika virus, wherein the vaccine or immunogenic composition isadministered as a first (prime) and a second (boost) administrationabout 1 to about 16 weeks apart and wherein the administration of thevaccine or immunogenic composition induces 14 and/or 28 days after theboost administration a seroconversion rate of at least 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% in a population of at least 20 seronegative human subjects,as determined by the plaque reduction neutralization test (PRNT).

Accordingly, certain aspects of the present disclosure relate to amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising (one) antigenfrom a Zika virus, wherein the vaccine or immunogenic composition isadministered as single dose or prime administration and wherein theadministration of the vaccine or immunogenic composition induces 14and/or 28 days after the single dose or prime administration aseroconversion rate of 25%, 30%, 40%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,or 90% in a population of at least 20 Zika virus seronegative humansubjects, as determined by the plaque reduction neutralization test(PRNT). The high seroconversion rate after single dose or primeadministration indicates an early onset of protection, which isbeneficial in an outbreak situation or a traveler visiting an endemicarea within a short period of time from the administration of thevaccine or immunogenic composition.

Accordingly, certain aspects of the present disclosure relate to amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising (one) antigenfrom a Zika virus, wherein the vaccine or immunogenic composition isadministered as single dose or prime administration and wherein theadministration of the vaccine or immunogenic composition induces 14and/or 28 days after the single dose or prime administration aseropositivity rate of 25%, 30%, 40%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,or 90% in a population of at least 20 Flavivirus naïve human subjects orof at least 20 Zika virus seronegative human subjects, as determined bythe plaque reduction neutralization test (PRNT). The high seroconversionrate after single dose or prime administration indicates an early onsetof protection, which is beneficial in an outbreak situation or atraveler visiting an endemic area within a short period of time from theadministration of the vaccine or immunogenic composition.

Certain other aspects of the present disclosure relate to a method oftreating or preventing, in particular preventing Zika virus infection ina human subject population in need thereof, comprising administering toindividual human subjects of said human subject population a vaccine orimmunogenic composition comprising (one) antigen from a Zika virus,wherein the vaccine or immunogenic composition is administered as asingle dose or as multiple doses as e.g. in a first (prime) and a second(boost) administration and wherein the administration of the vaccine orimmunogenic composition until 7 days after the administration inducessystemic side effects in less than 50% of a human subject population ofat least 20 human subjects, in particular in a population of at least 20flavivirus naïve human subjects or in particular in a population of atleast 20 Zika virus seronegative human subjects. The above methods areto be understood to also relate to corresponding uses of a vaccine orimmunogenic composition comprising (one) antigen from a Zika virus asdisclosed herein for the manufacture of a medicament for the treating orpreventing Zika virus infection.

Within the meaning of this disclosure “systemic side effects” are inparticular fever, headache, fatigue, arthralgia, myalgia and malaise.

The above methods are to be understood to also relate to a vaccine orimmunogenic composition comprising (one) antigen from a Zika virus asdisclosed herein for use in treating or preventing Zika virus infection.

In some embodiments the administration is intramuscular or subcutaneous.In some embodiments the administration includes the administration oftwo doses of the vaccine or immunogenic composition as described herein(e.g. 10 μg purified inactivated whole virus, such as a Zika virus witha mutation which is a tryptophan to glycine substitution at position 98of SEQ ID NO:1 or at a position corresponding to position 98 of SEQ IDNO: 1 as described herein) given about 1 to about 16 weeks apart (first(prime) and a second (boost) administration). In certain suchembodiments the Zika virus is a plaque purified clonal Zika virusisolate, in particular as described herein.

The above aspects of the present disclosure relate to the vaccines orimmunogenic compositions as described herein for use in treating orpreventing Zika virus infection in a human subject in need thereof, foruse in inducing an immune response in a human subject in need thereofand for use in preventing Zika virus disease in a human subject, fetusor newborn in need thereof. In some embodiments the administration isintramuscular or subcutaneous. In some embodiments the administrationincludes the administration of two doses of the vaccine or immunogeniccomposition as described herein (e.g. 10 μg purified inactivated wholevirus, such as a Zika virus with a mutation which is a tryptophan toglycine substitution at position 98 of SEQ ID NO:1 or at a positioncorresponding to position 98 of SEQ ID NO: 1 as described herein) givenabout 1 to about 16 weeks apart (first (prime) and a second (boost)administration). In certain such embodiments the Zika virus is a plaquepurified clonal Zika virus isolate, in particular as described herein.

The above aspects of the present disclosure relate to the use of thevaccines or immunogenic compositions as described herein in themanufacture of a medicament for treating or preventing Zika virusinfection in a human subject in need thereof for inducing an immuneresponse in a human subject in need thereof and in preventing Zika virusdisease in a human subject, fetus or newborn in need thereof. In someembodiments the administration is intramuscular or subcutaneous. In someembodiments the administration includes the administration of two dosesof the vaccine or immunogenic composition as described herein (e.g. 10μg purified inactivated whole virus, such as a Zika virus with amutation which is a tryptophan to glycine substitution at position 98 ofSEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO:1 as described herein) given about 1 to about 16 weeks apart (first(prime) and a second (boost) administration). In certain suchembodiments the Zika virus is a plaque purified clonal Zika virusisolate, in particular as described herein.

In some embodiments that may be combined with any of the precedingembodiments, the human subject is pregnant or intends to become pregnantor is a woman of childbearing potential.

In some embodiments that may be combined with any of the precedingembodiments, administration of the vaccine or immunogenic compositioninduces a protective immune response in the human subject. In someembodiments, the protective immune response induced in the human subjectis greater than a protective immune response induced in a correspondinghuman subject administered a vaccine or immunogenic compositioncontaining one or more antigens from a Zika virus lacking the at leastone non-human cell adaptation mutation. In some embodiments that may becombined with any of the preceding embodiments, administration of thevaccine or immunogenic composition induces the generation ofneutralizing antibodies to Zika virus in the human subject. In someembodiments, the concentration of neutralizing antibodies generated inthe human subject is higher than a concentration of neutralizingantibodies generated in a corresponding human subject administered avaccine or immunogenic composition comprising one or more antigens froma Zika virus lacking the at least one non-human cell adaptationmutation.

In some embodiments that may be combined with any of the precedingembodiments, the vaccine or immunogenic composition is administered by aroute selected from subcutaneous administration, transcutaneousadministration, intradermal administration, subdermal administration,intramuscular administration, peroral administration, intranasaladministration, buccal administration, intraperitoneal administration,intravaginal administration, anal administration and/or intracranialadministration. In some embodiments that may be combined with any of thepreceding embodiments, the vaccine or immunogenic composition isadministered one or more times. In some embodiments, the vaccine orimmunogenic composition is administered as a first (prime) and a second(boost) administration. In some embodiments, the second (boost)administration is administered at least 28 days after the first (prime)administration. In some embodiments the administration includes theadministration of two doses of the vaccine or immunogenic composition asdescribed herein (e.g. 10 μg purified inactivated whole virus, such as aZika virus with a mutation which is a tryptophan to glycine substitutionat position 98 of SEQ ID NO:1 or at a position corresponding to position98 of SEQ ID NO: 1 as described herein) given about 1 to about 16 weeksapart (first (prime) and a second (boost) administration). In certainsuch embodiments the Zika virus is a plaque purified clonal Zika virusisolate.

In some embodiments that may be combined with any of the precedingembodiments, the virus preparation is mixed with an adjuvant. In someembodiments, the adjuvant is selected from aluminum salts, toll-likereceptor (TLR) agonists, monophosphoryl lipid A (MLA), synthetic lipidA, lipid A mimetics or analogs, MLA derivatives, cytokines, saponins,muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide(LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes,cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamerparticles, microparticles, liposomes, Complete Freund's Adjuvant (CFA),and/or Incomplete Freund's Adjuvant (IFA). In some embodiments, theadjuvant is an aluminum salt. In some embodiments, the adjuvant isselected from alum, aluminum phosphate, aluminum hydroxide, potassiumaluminum sulfate, and/or Alhydrogel 85. In some embodiments, at least75%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% of one or more antigens in thevirus preparation are adsorbed to the adjuvant.

In some embodiments that may be combined with any of the precedingembodiments, the at least one non-human cell adaptation mutation is inZika virus NS1. In some embodiments, the at least one adaptationmutation occurs at position 98 of SEQ ID NO: 1, or at a positioncorresponding to position 98 of SEQ ID NO: 1. In some embodiments, theat least one adaptation mutation is a Trp98Gly mutation. In someembodiments that may be combined with any of the preceding embodiments,the at least one adaptation mutation enhances genetic stability ascompared to a Zika virus lacking the at least one adaptation mutation.In some embodiments that may be combined with any of the precedingembodiments, the at least one adaptation mutation enhances viralreplication as compared to a Zika virus lacking the at least oneadaptation mutation. In some embodiments that may be combined with anyof the preceding embodiments, the Zika virus does not comprise amutation in Envelope protein E (Env).

In some embodiments that may be combined with any of the precedingembodiments, the population of Zika viruses is heterogeneous. In someembodiments that may be combined with any of the preceding embodiments,the population of Zika viruses comprises a Zika virus clinical isolate.In some embodiments, the Zika virus clinical isolate is from strainPRVABC59. In some embodiments that may be combined with any of thepreceding embodiments, the population of Zika viruses comprises a Zikavirus that has been previously passaged one or more times in cellculture. In some embodiments that may be combined with any of thepreceding embodiments, the inoculum comprises human serum. In someembodiments that may be combined with any of the preceding embodiments,the inoculum comprises one or more adventitious agents. In someembodiments, the Zika virus clonal isolate is substantially free of theone or more adventitious agents.

In some embodiments that may be combined with any of the precedingembodiments, the methods further include one or more additional plaquepurifications of the Zika virus clonal isolate. In some embodiments, theZika virus clonal isolate is further plaque purified two or more times.In some embodiments that may be combined with any of the precedingembodiments, the methods further include passaging the Zika virus clonalisolate one or more times in cell culture. In some embodiments, the Zikavirus clonal isolate is passaged two or more times.

In some embodiments that may be combined with any of the precedingembodiments, the methods further include formulating a vaccine orimmunogenic composition comprising one or more antigens from the Zikavirus clonal isolate. In some embodiments, the vaccine or immunogeniccomposition is a purified antigen vaccine or immunogenic composition, asubunit vaccine or immunogenic composition, an inactivated whole virusvaccine or immunogenic composition, or an attenuated virus vaccine orimmunogenic composition. In some embodiments, the vaccine or immunogeniccomposition is a purified inactivated whole virus vaccine or immunogeniccomposition. In some embodiments, the Zika virus clonal isolate waschemically inactivated. In some embodiments, the Zika virus clonalisolate was chemically inactivated with one or more of a detergent,formalin, beta-propiolactone (BPL), binary ethylamine (BEI), acetylethyleneimine, methylene blue, and psoralen. In some embodiments, theZika virus clonal isolate was chemically inactivated with formalin.

In some embodiments that may be combined with any of the precedingembodiments, the methods further include admixing the vaccine orimmunogenic composition with an adjuvant. In some embodiments, theadjuvant is selected from aluminum salts, toll-like receptor (TLR)agonists, monophosphoryl lipid A (MLA), synthetic lipid A, lipid Amimetics or analogs, MLA derivatives, cytokines, saponins, muramyldipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) ofgram-negative bacteria, polyphosphazenes, emulsions, virosomes,cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamerparticles, microparticles, liposomes, Complete Freund's Adjuvant (CFA),and Incomplete Freund's Adjuvant (IFA). In some embodiments, theadjuvant is an aluminum salt. In some embodiments, the adjuvant isselected from alum, aluminum phosphate, aluminum hydroxide, potassiumaluminum sulfate, and Alhydrogel 85. In some embodiments, at least 75%,at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% of the one or more antigens areadsorbed to the adjuvant. In some embodiments that may be combined withany of the preceding embodiments, the vaccine or immunogenic compositioncomprises 1 μg to about 40 μg of the purified inactivated whole virus,or 1 μg to about 30 μg of the purified inactivated whole virus, or 1 μgto about 20 μg of the purified inactivated whole virus, in particular 2μg, or 5 μg, or 10 μg, or 15 μg or 20 μg or in particular 10 μg purifiedinactivated whole virus, such as a Zika virus with a mutation which is atryptophan to glycine substitution at position 98 of SEQ ID NO:1 or at aposition corresponding to position 98 of SEQ ID NO: 1 as describedherein. In some embodiments, the vaccine or immunogenic compositioncomprises 1 μg to about 30 μg of the purified inactivated whole virus,in particular 2 μg, 5 μg or 10 μg, or in particular 10 μg of a purifiedinactivated whole Zika virus comprising a Trp98Gly mutation at position98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQID NO:1, wherein the Zika virus is derived from strain PRVABC59. In someembodiments, the vaccine or immunogenic composition comprises 1 μg toabout 30 μg of the purified inactivated whole virus, in particular 2 μg,5 μg or 10 μg, or in particular 10 μg of a purified inactivated wholeZika virus comprising a Trp98Gly mutation at position 98 of SEQ ID NO:1, or at a position corresponding to position 98 of SEQ ID NO:1, whereinthe Zika virus is derived from strain PRVABC59 comprising the genomicsequence according to SEQ ID NO:2.

In some embodiments that may be combined with any of the precedingembodiments, the vaccine or immunogenic composition comprises from about0.1 μg Env to about 100 μg Env. In certain such embodiments the Zikavirus is a plaque purified clonal Zika virus isolate. In someembodiments, the vaccine or immunogenic composition is unadjuvanted.

In some embodiments that may be combined with any of the precedingembodiments, the Zika virus clonal isolate is a homogenous geneticpopulation. In some embodiments, the Zika virus clonal isolate does notcontain a mutation in Envelope protein E (Env). In some embodiments, theZika virus clonal isolate contains at least one mutation. In someembodiments, the at least one mutation is in Zika virus Non-structuralprotein 1 (NS1). In some embodiments, the at least one mutation occursat position 98 of SEQ ID NO: 1, or at a position corresponding toposition 98 of SEQ ID NO: 1. In some embodiments, the at least onemutation is a Trp98Gly mutation. In some embodiments the at least onemutation is not in Envelope protein E (Env). In some embodiments, the atleast one mutation enhances genetic stability as compared to a Zikavirus lacking the at least one mutation. In some embodiments, the atleast one mutation enhances viral replication as compared to a Zikavirus lacking the at least one mutation.

Other aspects of the present disclosure relate to a vaccine orimmunogenic composition containing one or more antigens from a plaquepurified clonal Zika virus isolate. In some embodiments, the plaquepurified clonal Zika virus isolate was plaque purified from cellscontacted with an inoculum comprising a population of Zika viruses. Insome embodiments, the cells are non-human cells. In some embodiments,the cells are insect cells. In some embodiments, the insect cells aremosquito cells. In some embodiments, the cells are mammalian cells. Insome embodiments, the mammalian cells are monkey cells. In someembodiments, the monkey cells are from a Vero cell line. In someembodiments, the Vero cell line is a WHO Vero 10-87 cell line.

In some embodiments that may be combined with any of the precedingembodiments, the population of Zika viruses was heterogeneous. In someembodiments that may be combined with any of the preceding embodiments,the population of Zika viruses comprised a Zika virus clinical isolate.In some embodiments, the Zika virus clinical isolate is from strainPRVABC59. In some embodiments that may be combined with any of thepreceding embodiments, the population of Zika viruses comprised a Zikavirus that had been previously passaged one or more times in cellculture. In some embodiments that may be combined with any of thepreceding embodiments, the inoculum comprised human serum. In someembodiments that may be combined with any of the preceding embodiments,the inoculum comprised one or more adventitious agents. In someembodiments, the plaque purified clonal Zika virus isolate issubstantially free of the one or more adventitious agents.

In some embodiments that may be combined with any of the precedingembodiments, the plaque purified clonal Zika virus isolate is modifiedas compared to a wild-type Zika virus. In some embodiments that may becombined with any of the preceding embodiments, the plaque purifiedclonal Zika virus isolate is a homogenous genetic population. In someembodiments that may be combined with any of the preceding embodiments,the plaque purified clonal Zika virus isolate does not include amutation in Envelope protein E (Env). In some embodiments that may becombined with any of the preceding embodiments, the plaque purifiedclonal Zika virus isolate comprises at least one mutation. In someembodiments, the at least one mutation is in Zika virus Non-structuralprotein 1 (NS1). In some embodiments, the at least one mutation occursat position 98 of SEQ ID NO: 1, or at a position corresponding toposition 98 of SEQ ID NO: 1. In some embodiments, the at least onemutation is a Trp98Gly mutation. In some embodiments, the at least onemutation is not in Zika virus Envelope protein E (Env). In someembodiments, the at least one mutation enhances genetic stability ascompared to a Zika virus lacking the at least one mutation. In someembodiments, the at least one mutation enhances viral replication ascompared to a Zika virus lacking the at least one mutation. In someembodiments that may be combined with any of the preceding embodiments,the plaque purified clonal Zika virus isolate is an African lineagevirus or an Asian lineage virus. In some embodiments, the plaquepurified clonal Zika virus isolate is an Asian lineage virus.

In some embodiments that may be combined with any of the precedingembodiments, the vaccine or immunogenic composition is a purifiedantigen vaccine or immunogenic composition, a subunit vaccine orimmunogenic composition, an inactivated whole virus vaccine orimmunogenic composition, or an attenuated virus vaccine or immunogeniccomposition. In some embodiments, the vaccine or immunogenic compositionis an inactivated whole virus vaccine or immunogenic composition. Insome embodiments that may be combined with any of the precedingembodiments, the plaque purified clonal Zika virus isolate waschemically inactivated. In some embodiments, the plaque purified Zikavirus was chemically inactivated with one or more of a detergent,formalin, beta-propiolactone (BPL), binary ethylamine (BEI), acetylethyleneimine, methylene blue, and psoralen. In some embodiments, theplaque purified clonal Zika virus isolate was chemically inactivatedwith formalin.

In some embodiments that may be combined with any of the precedingembodiments, the vaccine or immunogenic composition further comprises anadjuvant. In some embodiments, the adjuvant is selected from aluminumsalts, toll-like receptor (TLR) agonists, monophosphoryl lipid A (MLA),synthetic lipid A, lipid A mimetics or analogs, MLA derivatives,cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos,lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes,emulsions, virosomes, cochleates, poly(lactide-co-glycolides) (PLG)microparticles, poloxamer particles, microparticles, liposomes, CompleteFreund's Adjuvant (CFA), and Incomplete Freund's Adjuvant (IFA). In someembodiments, the adjuvant is an aluminum salt. In some embodiments, theadjuvant is selected from alum, aluminum phosphate, aluminum hydroxide,potassium aluminum sulfate, and Alhydrogel 85. In some embodiments, atleast 75%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% of the one or moreantigens are adsorbed to the adjuvant. In some embodiments that may becombined with any of the preceding embodiments, the vaccine orimmunogenic composition contains from 0.1 μg to about 25 μg of thepurified inactivated whole virus, such as a Zika virus with a mutationwhich is a tryptophan to glycine substitution at position 98 of SEQ IDNO:1 as described herein, in particular 2 μg, 5 μg or 10 μg, or inparticular 10 μg purified inactivated whole virus or about 0.1 μg Env toabout 100 μg Env. In certain such embodiments the Zika virus is a plaquepurified clonal Zika virus isolate. In some embodiments, the vaccine orimmunogenic composition is unadjuvanted.

It is to be understood that one, some, or all of the properties of thevarious embodiments described above and herein may be combined to formother embodiments of the present disclosure. These and other aspects ofthe present disclosure will become apparent to one of skill in the art.These and other embodiments of the present disclosure are furtherdescribed by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows bright field microscopy images of Vero cell monolayers mockinfected (top) or infected with ZIKAV strain PRVABC59 (bottom).

FIG. 2 shows growth kinetics of ZIKAV PRVABC59 P1 on Vero cellmonolayers, as determined by TCID50.

FIG. 3 shows potency assay testing (TCID50) of Zika virus PRVABC59 P5clones a-f.

FIG. 4 shows bright-field microscopy images depicting the cytopathiceffect (CPE) of growth of Zika virus PRVABC59 P6 clones a-f on Vero cellmonolayers.

FIG. 5 shows potency assay testing (TCID50) of Zika virus PRVABC59 P6clones a-f

FIG. 6 shows an amino acid sequence alignment comparing the envelopeglycoprotein sequence of Zika virus near residue 330 from Zika virusstrains PRVABC59 P6e (SEQ ID NO: 8) and PRVABC59 (SEQ ID NO: 9) withseveral other flaviviruses (WNV (SEQ ID NO: 10); JEV (SEQ ID NO: 11);SLEV (SEQ ID NO: 12); YFV (SEQ ID NO: 13); DENY 1 16007 (SEQ ID NO: 14);DENY 2 16681 (SEQ ID NO: 15); DENY 3 16562 (SEQ ID NO: 16); and DENY 41036 (SEQ ID NO: 17)).

FIG. 7 shows an amino acid sequence alignment comparing the NS1 proteinsequence of Zika virus near residue 98 from Zika virus strains PRVABC59P6e (SEQ ID NO: 18) and PRVABC59 (SEQ ID NO: 19) with several otherflaviviruses (WNV (SEQ ID NO: 20); JEV (SEQ ID NO: 21); SLEV (SEQ ID NO:22); YFV (SEQ ID NO: 23); DENY 1 16007 (SEQ ID NO: 24); DENY 2 16681(SEQ ID NO: 25); DENY 3 16562 (SEQ ID NO: 26); and DENY 4 1036 (SEQ IDNO: 27)).

FIG. 8 shows the plaque phenotype of ZIKAV PRVABC59 P6 virus clones a-fcompared to ZIKAV PRVABC59 P1 virus.

FIG. 9 shows the mean plaque size of ZIKAV PRVABC59 P6 virus clonescompared to ZIKAV PRVABC59 P1 virus.

FIG. 10 shows the growth kinetics of ZIKAV PRVABC59 P6 clones a-f inVero cells under serum-free growth conditions.

FIG. 11 shows a schematic of the steps taken to prepare PRVABC59 P6b andP6e formulated drug product for the immunization experiments.

FIG. 12A shows the schedule of dosing of CD-1 mice with vaccineformulations derived from the ZIKAV PRVABC59 P6b and P6e clones. PBS wasused as placebo.

FIG. 12B shows the serum ZIKAV neutralizing antibody titers of CD-1 miceimmunized as described in FIG. 12A using vaccine formulations derivedfrom ZIKAV PRVABC59 P6b and P6e clones. ZIKAV neutralizing antibodytiters were determined by Reporter Virus Particle (RVP) neutralizationassay. Solid lines represent the geometric mean of a group. The limit ofdetection (1.93 log₁₀) is represented by a dashed line.

FIG. 13A shows the schedule of dosing of AG129 mice with vaccineformulations derived from the ZIKAV PRVABC59 P6b and P6e clones. PBS wasused as a placebo.

FIG. 13B shows the serum ZIKAV neutralizing antibody titers of AG129mice immunized as described in FIG. 13A using vaccine formulationsderived from ZIKAV PRVABC59 P6b and P6e clones. Solid lines representthe geometric mean of a group. The limit of detection (1.30 log₁₀) isrepresented by a dashed line. Animals with no detectable titer (<1.30)were assigned a titer of 0.5.

FIG. 14 shows the mean weight of AG129 test groups post-challenge,represented as a percentage of starting weight. Error bars representstandard deviation.

FIG. 15 shows the serum viremia of individual AG129 mice two dayspost-challenge, reported as PFU/mL. Solid lines represent the mean of agroup. The limit of detection (2.0 log₁₀) is represented by a dashedline.

FIG. 16 shows the survival analysis of AG129 test groups post-challenge.

FIG. 17 shows the pre-challenge serum circulating ZIKAV neutralizingantibody (Nab) titers following passive transfer of pooled sera fromvaccinated and challenged AG129 mice.

FIG. 18 shows the mean body weight of passive transfer and control micechallenged with Zika virus.

FIG. 19 shows the serum viremia of individual AG129 mice three dayspost-challenge, reported as PFU/mL.

FIG. 20 shows the survival analysis of passive transfer and control micechallenged with Zika virus.

FIG. 21 shows the correlation between ZIKAV neutralizing antibody titersand viremia observed in passive transfer mice.

FIG. 22 shows the survival analysis of AG129 mice after challenge withpreMVS stocks of P6a and P6e using a Kaplan Meier survival curve.

FIG. 23 shows the mean body weight as expressed in percentage ofstarting weight at time of invention after challenge with preMVS stocksof P6a and P6e. The dashed line represents 100% of starting weight forreference.

FIG. 24 shows the serum viremia of individual AG129 mice three dayspost-challenge with preMVS stocks of P6a and P6e, reported as PFU/mL.The dashed line represents the limit of detection of the assay.

FIG. 25 shows compiled kinetics of inactivation data. Data comparesinfectious potency (TCID50) to RNA copy, and completeness ofinactivation (COI) for samples from the four toxicology lots. These dataindicate that the sensitivity of the COI assay is greater than TCID50.

FIG. 26 shows a comparison of C6/36 and Vero sensitivity in the assay asdemonstrated with an input virus titer of 0.31 TCID50.

FIG. 27 shows a logistic regression analysis of CPE vs. log TCID50 usingC6/36 cells site that include 99% confidence intervals around a targetvalue of 0.01 TCID50/well (−2 log TCID50/well); the model predicts 0.85%of wells will be positive.

FIG. 28 shows chromatograms of PBS (a) and PBS solutions containing0.049 μg/mL (b), 0.098 μg/mL (c), 0.196 μg/mL (d), 0.491 μg/mL (e),0.982 μg/mL (f), and 1.964 μg/mL (g) formaldehyde.

FIG. 29 shows a summary of the Clinical Study Design for Example 6.

FIG. 30 shows the Geometric Mean Titers (GMTs) determined using PRNT ofthe Human subjects in Example 6

FIG. 31 shows the percentage of human subjects achieving seroconversiondetermined using PRNT at Day 29 (day 28 after prime dose) and Day 57 (28days after boost dose) of the study described in Example 6.

FIG. 32 shows the plot of the percentage of human subjects achieving aparticular Geometric Mean Titer (determined using PRNT) on day 29 (day28 after prime dose) of the study described in Example 6.

FIG. 33 shows the plot of the percentage of human subjects achieving aparticular Geometric Mean Titer (determined using PRNT) on day 57 (day56 after prime dose) of the study described in Example 6.

DETAILED DESCRIPTION General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3d edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., (2003)); the seriesMethods in Enzymology (Academic Press, Inc.): PCR 2: A PracticalApproach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Antibodies, A Laboratory Manual (Harlow and Lane, eds. (1988), andAnimal Cell Culture (R. I. Freshney, ed. (1987)); OligonucleotideSynthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology (J. M.Walker, ed. Humana Press (1983)); Cell Biology: A Laboratory Notebook(J. E. Celis, ed., Academic Press (1998)) Academic Press; Animal CellCulture (R. I. Freshney), ed., 1987); Introduction to Cell and TissueCulture (J. P. Mather and P. E. Roberts, eds. Plenum Press (1998)); Celland Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths,and D. G. Newell, eds., J. Wiley and Sons (1993-8)); Handbook ofExperimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., Cold Spring Harbor Laboratory (1987)); PCR: The Polymerase ChainReaction, (Mullis et al., eds., Springer (1994)); Current Protocols inImmunology (J. E. Coligan et al., eds., Wiley (1991)); Short Protocolsin Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.Janeway and P. Travers, (1997)); Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press,(1988-1989)); Monoclonal Antibodies: A Practical Approach (P. Shepherdand C. Dean, eds., Oxford University Press, (2000)); Using Antibodies: ALaboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor LaboratoryPress, (1999)); and The Antibodies (M. Zanetti and J. D. Capra, eds.,Harwood Academic Publishers, (1995)).

Zika Virus

Certain aspects of the present disclosure relate to at least one Zikavirus (e.g., a Zika virus clonal isolate, a Zika virus purified by themethods described herein, a Zika virus comprising one or more non-humancell adaptation mutations, etc.) that may be useful in vaccines and/orimmunogenic compositions including, without limitation, purifiedviruses, inactivated viruses, attenuated viruses, recombinant viruses,or purified and/or recombinant viral proteins for subunit vaccines.

Zika virus (ZIKV) is a mosquito-borne flavivirus first isolated from asentinel rhesus monkey in the Zika Forest in Uganda in 1947. Since thattime, isolations have been made from humans in both Africa and Asia, andmore recently, the Americas. ZIKV is found in two (possibly three)lineages: an African lineage (possibly separate East and West Africanlineages) and an Asian lineage. Accordingly, examples of suitable Zikaviruses of the present disclosure include, without limitation, virusesfrom the African and/or Asian lineages. In some embodiments, the Zikavirus is an African lineage virus. In some embodiments, the Zika virusis an Asian lineage virus. Additionally, multiple strains within theAfrican and Asian lineages of Zika virus have been previouslyidentified. Any one or more suitable strains of Zika virus known in theart may be used in the present disclosure, including, for examples,strains Mr 766, ArD 41519, IbH 30656, P6-740, EC Yap, FSS13025, ArD7117, ArD 9957, ArD 30101, ArD 30156, ArD 30332, HD 78788, ArD 127707,ArD 127710, ArD 127984, ArD 127988, ArD 127994, ArD 128000, ArD 132912,132915, ArD 141170, ArD 142623, ArD 149917, ArD 149810, ArD 149938, ArD157995, ArD 158084, ArD 165522, ArD 165531, ArA 1465, ArA 27101, ArA27290, ArA 27106, ArA 27096, ArA 27407, ArA 27433, ArA 506/96, ArA975-99, Ara 982-99, ArA 986-99, ArA 2718, ArB 1362, Nigeria68,Malaysia66, Kedougou84, Suriname, MR1429, PRVABC59, ECMN2007,DakAr41524, H/PF/2013, R103451, 103344, 8375, JMB-185, ZIKV/H,sapiens/Brazil/Natal/2015, SPH2015, ZIKV/Hu/Chiba/S36/2016, and/orCuba2017. In some embodiments, strain PRVABC59 is used in the presentdisclosure.

In some embodiments, an example of a Zika virus genome sequence is setforth below as SEQ ID NO: 2:

    1 gttgttgatc tgtgtgaatc agactgcgac agttcgagtt tgaagcgaaa gctagcaaca    61 gtatcaacag gttttatttt ggatttggaa acgagagttt ctggtcatga aaaacccaaa   121 aaagaaatcc ggaggattcc ggattgtcaa tatgctaaaa cgcggagtag cccgtgtgag   181 cccctttggg ggcttgaaga ggctgccagc cggacttctg ctgggtcatg ggcccatcag   241 gatggtcttg gcgattctag cctttttgag attcacggca atcaagccat cactgggtct   301 catcaataga tggggttcag tggggaaaaa agaggctatg gaaacaataa agaagttcaa   361 gaaagatctg gctgccatgc tgagaataat caatgctagg aaggagaaga agagacgagg   421 cgcagatact agtgtcggaa ttgttggcct cctgctgacc acagctatgg cagcggaggt   481 cactagacgt gggagtgcat actatatgta cttggacaga aacgatgctg gggaggccat   541 atcttttcca accacattgg ggatgaataa gtgttatata cagatcatgg atcttggaca   601 catgtgtgat gccaccatga gctatgaatg ccctatgctg gatgaggggg tggaaccaga   661 tgacgtcgat tgttggtgca acacgacgtc aacttgggtt gtgtacggaa cctgccatca   721 caaaaaaggt gaagcacgga gatctagaag agctgtgacg ctcccctccc attccaccag   781 gaagctgcaa acgcggtcgc aaacctggtt ggaatcaaga gaatacacaa agcacttgat   841 tagagtcgaa aattggatat tcaggaaccc tggcttcgcg ttagcagcag ctgccatcgc   901 ttggcttttg ggaagctcaa cgagccaaaa agtcatatac ttggtcatga tactgctgat   961 tgccccggca tacagcatca ggtgcatagg agtcagcaat agggactttg tggaaggtat  1021 gtcaggtggg acttgggttg atgttgtctt ggaacatgga ggttgtgtca ccgtaatggc  1081 acaggacaaa ccgactgtcg acatagagct ggttacaaca acagtcagca acatggcgga  1141 ggtaagatcc tactgctatg aggcatcaat atcagacatg gcttctgaca gccgctgccc  1201 aacacaaggt gaagcctacc ttgacaagca atcagacact caatatgtct gcaaaagaac  1261 gttagtggac agaggctggg gaaatggatg tggacttttt ggcaaaggga gcctggtgac  1321 atgcgctaag tttgcatgct ccaagaaaat gaccgggaag agcatccagc cagagaatct  1381 ggagtaccgg ataatgctgt cagttcatgg ctcccagcac agtgggatga tcgttaatga  1441 cacaggacat gaaactgatg agaatagagc gaaagttgag ataacgccca attcaccgag  1501 agccgaagcc accctggggg gttttggaag cctaggactt gattgtgaac cgaggacagg  1561 ccttgacttt tcagatttgt attacttgac tatgaataac aagcactggt tggttcacaa  1621 ggagtggttc cacgacattc cattaccttg gcacgctggg gcagacaccg gaactccaca  1681 ctggaacaac aaagaagcac tggtagagtt caaggacgca catgccaaaa ggcaaactgt  1741 cgtggttcta gggagtcaag aaggagcagt tcacacggcc cttgctggag ctctggaggc  1801 tgagatggat ggtgcaaagg gaaggctgtc ctctggccac ttgaaatgtc gcctgaaaat  1861 ggataaactt agattgaagg gcgtgtcata ctccttgtgt actgcagcgt tcacattcac  1921 caagatcccg gctgaaacac tgcacgggac agtcacagtg gaggtacagt acgcagggac  1981 agatggacct tgcaaggttc cagctcagat ggcggtggac atgcaaactc tgaccccagt  2041 tgggaggttg ataaccgcta accccgtaat cactgaaagc actgagaact ctaagatgat  2101 gctggaactt gatccaccat ttggggactc ttacattgtc ataggagtcg gggagaagaa  2161 gatcacccac cactggcaca ggagtggcag caccattgga aaagcatttg aagccactgt  2221 gagaggtgcc aagagaatgg cagtcttggg agacacagcc tgggactttg gatcagttgg  2281 aggcgctctc aactcattgg gcaagggcat ccatcaaatt tttggagcag ctttcaaatc  2341 attgtttgga ggaatgtcct ggttctcaca aattctcatt ggaacgttgc tgatgtggtt  2401 gggtctgaac acaaagaatg gatctatttc ccttatgtgc ttggccttag ggggagtgtt  2461 gatcttctta tccacagccg tctctgctga tgtggggtgc tcggtggact tctcaaagaa  2521 ggagacgaga tgcggtacag gggtgttcgt ctataacgac gttgaagcct ggagggacag  2581 gtacaagtac catcctgact ccccccgtag attggcagca gcagtcaagc aagcctggga  2641 agatggtatc tgcgggatct cctctgtttc aagaatggaa aacatcatgt ggagatcagt  2701 agaaggggag ctcaacgcaa tcctggaaga gaatggagtt caactgacgg tcgttgtggg  2761 atctgtaaaa aaccccatgt ggagaggtcc acagagattg cccgtgcctg tgaacgagct  2821 gccccacggc tggaaggctt gggggaaatc gtatttcgtc agagcagcaa agacaaataa  2881 cagctttgtc gtggatggtg acacactgaa ggaatgccca ctcaaacata gagcatggaa  2941 cagctttctt gtggaggatc atgggttcgg ggtatttcac actagtgtct ggctcaaggt  3001 tagagaagat tattcattag agtgtgatcc agccgttatt ggaacagctg ttaagggaaa  3061 ggaggctgta cacagtgatc taggctactg gattgagagt gagaagaatg acacatggag  3121 gctgaagagg gcccatctga tcgagatgaa aacatgtgaa tggccaaagt cccacacatt  3181 gtggacagat ggaatagaag agagtgatct gatcataccc aagtctttag ctgggccact  3241 cagccatcac aataccagag agggctacag gacccaaatg aaagggccat ggcacagtga  3301 agagcttgaa attcggtttg aggaatgccc aggcactaag gtccacgtgg aggaaacatg  3361 tggaacaaga ggaccatctc tgagatcaac cactgcaagc ggaagggtga tcgaggaatg  3421 gtgctgcagg gagtgcacaa tgcccccact gtcgttccgg gctaaagatg gctgttggta  3481 tggaatggag ataaggccca ggaaagaacc agaaagcaac ttagtaaggt caatggtgac  3541 tgcaggatca actgatcaca tggaccactt ctcccttgga gtgcttgtga tcctgctcat  3601 ggtgcaggaa gggctgaaga agagaatgac cacaaagatc atcataagca catcaatggc  3661 agtgctggta gctatgatcc tgggaggatt ttcaatgagt gacctggcta agcttgcaat  3721 tttgatgggt gccaccttcg cggaaatgaa cactggagga gatgtagctc atctggcgct  3781 gatagcggca ttcaaagtca gaccagcgtt gctggtatct ttcatcttca gagctaattg  3841 gacaccccgt gaaagcatgc tgctggcctt ggcctcgtgt cttttgcaaa ctgcgatctc  3901 cgccttggaa ggcgacctga tggttctcat caatggtttt gctttggcct ggttggcaat  3961 acgagcgatg gttgttccac gcactgataa catcaccttg gcaatcctgg ctgctctgac  4021 accactggcc cggggcacac tgcttgtggc gtggagagca ggccttgcta cttgcggggg  4081 gtttatgctc ctctctctga agggaaaagg cagtgtgaag aagaacttac catttgtcat  4141 ggccctggga ctaaccgctg tgaggctggt cgaccccatc aacgtggtgg gactgctgtt  4201 gctcacaagg agtgggaagc ggagctggcc ccctagcgaa gtactcacag ctgttggcct  4261 gatatgcgca ttggctggag ggttcgccaa ggcagatata gagatggctg ggcccatggc  4321 cgcggtcggt ctgctaattg tcagttacgt ggtctcagga aagagtgtgg acatgtacat  4381 tgaaagagca ggtgacatca catgggaaaa agatgcggaa gtcactggaa acagtccccg  4441 gctcgatgtg gcgctagatg agagtggtga tttctccctg gtggaggatg acggtccccc  4501 catgagagag atcatactca aggtggtcct gatgaccatc tgtggcatga acccaatagc  4561 catacccttt gcagctggag cgtggtacgt atacgtgaag actggaaaaa ggagtggtgc  4621 tctatgggat gtgcctgctc ccaaggaagt aaaaaagggg gagaccacag atggagtgta  4681 cagagtaatg actcgtagac tgctaggttc aacacaagtt ggagtgggag ttatgcaaga  4741 gggggtcttt cacactatgt ggcacgtcac aaaaggatcc gcgctgagaa gcggtgaagg  4801 gagacttgat ccatactggg gagatgtcaa gcaggatctg gtgtcatact gtggtccatg  4861 gaagctagat gccgcctggg atgggcacag cgaggtgcag ctcttggccg tgccccccgg  4921 agagagagcg aggaacatcc agactctgcc cggaatattt aagacaaagg atggggacat  4981 tggagcggtt gcgctggatt acccagcagg aacttcagga tctccaatcc tagacaagtg  5041 tgggagagtg ataggacttt atggcaatgg ggtcgtgatc aaaaacggga gttatgttag  5101 tgccatcacc caagggagga gggaggaaga gactcctgtt gagtgcttcg agccctcgat  5161 gctgaagaag aagcagctaa ctgtcttaga cttgcatcct ggagctggga aaaccaggag  5221 agttcttcct gaaatagtcc gtgaagccat aaaaacaaga ctccgtactg tgatcttagc  5281 tccaaccagg gttgtcgctg ctgaaatgga ggaggccctt agagggcttc cagtgcgtta  5341 tatgacaaca gcagtcaatg tcacccactc tggaacagaa atcgtcgact taatgtgcca  5401 tgccaccttc acttcacgtc tactacagcc aatcagagtc cccaactata atctgtatat  5461 tatggatgag gcccacttca cagatccctc aagtatagca gcaagaggat acatttcaac  5521 aagggttgag atgggcgagg cggctgccat cttcatgacc gccacgccac caggaacccg  5581 tgacgcattt ccggactcca actcaccaat tatggacacc gaagtggaag tcccagagag  5641 agcctggagc tcaggctttg attgggtgac ggatcattct ggaaaaacag tttggtttgt  5701 tccaagcgtg aggaacggca atgagatcgc agcttgtctg acaaaggctg gaaaacgggt  5761 catacagctc agcagaaaga cttttgagac agagttccag aaaacaaaac atcaagagtg  5821 ggactttgtc gtgacaactg acatttcaga gatgggcgcc aactttaaag ctgaccgtgt  5881 catagattcc aggagatgcc taaagccggt catacttgat ggcgagagag tcattctggc  5941 tggacccatg cctgtcacac atgccagcgc tgcccagagg agggggcgca taggcaggaa  6001 tcccaacaaa cctggagatg agtatctgta tggaggtggg tgcgcagaga ctgacgaaga  6061 ccatgcacac tggcttgaag caagaatgct ccttgacaat atttacctcc aagatggcct  6121 catagcctcg ctctatcgac ctgaggccga caaagtagca gccattgagg gagagttcaa  6181 gcttaggacg gagcaaagga agacctttgt ggaactcatg aaaagaggag atcttcctgt  6241 ttggctggcc tatcaggttg catctgccgg aataacctac acagatagaa gatggtgctt  6301 tgatggcacg accaacaaca ccataatgga agacagtgtg ccggcagagg tgtggaccag  6361 acacggagag aaaagagtgc tcaaaccgag gtggatggac gccagagttt gttcagatca  6421 tgcggccctg aagtcattca aggagtttgc cgctgggaaa agaggagcgg cttttggagt  6481 gatggaagcc ctgggaacac tgccaggaca catgacagag agattccagg aagccattga  6541 caacctcgct gtgctcatgc gggcagagac tggaagcagg ccttacaaag ccgcggcggc  6601 ccaattgccg gagaccctag agaccataat gcttttgggg ttgctgggaa cagtctcgct  6661 gggaatcttc ttcgtcttga tgaggaacaa gggcataggg aagatgggct ttggaatggt  6721 gactcttggg gccagcgcat ggctcatgtg gctctcggaa attgagccag ccagaattgc  6781 atgtgtcctc attgttgtgt tcctattgct ggtggtgctc atacctgagc cagaaaagca  6841 aagatctccc caggacaacc aaatggcaat catcatcatg gtagcagtag gtcttctggg  6901 cttgattacc gccaatgaac tcggatggtt ggagagaaca aagagtgacc taagccatct  6961 aatgggaagg agagaggagg gggcaaccat aggattctca atggacattg acctgcggcc  7021 agcctcagct tgggccatct atgctgcctt gacaactttc attaccccag ccgtccaaca  7081 tgcagtgacc acctcataca acaactactc cttaatggcg atggccacgc aagctggagt  7141 gttgtttggc atgggcaaag ggatgccatt ctacgcatgg gactttggag tcccgctgct  7201 aatgataggt tgctactcac aattaacacc cctgacccta atagtggcca tcattttgct  7261 cgtggcgcac tacatgtact tgatcccagg gctgcaggca gcagctgcgc gtgctgccca  7321 gaagagaacg gcagctggca tcatgaagaa ccctgttgtg gatggaatag tggtgactga  7381 cattgacaca atgacaattg acccccaagt ggagaaaaag atgggacagg tgctactcat  7441 agcagtagcc gtctccagcg ccatactgtc gcggaccgcc tgggggtggg gggaggctgg  7501 ggctctgatc acagccgcaa cttccacttt gtgggaaggc tctccgaaca agtactggaa  7561 ctcctctaca gccacttcac tgtgtaacat ttttagggga agttacttgg ctggagcttc  7621 tctaatctac acagtaacaa gaaacgctgg cttggtcaag agacgtgggg gtggaacagg  7681 agagaccctg ggagagaaat ggaaggcccg cttgaaccag atgtcggccc tggagttcta  7741 ctcctacaaa aagtcaggca tcaccgaggt gtgcagagaa gaggcccgcc gcgccctcaa  7801 ggacggtgtg gcaacgggag gccatgctgt gtcccgagga agtgcaaagc tgagatggtt  7861 ggtggagcgg ggatacctgc agccctatgg aaaggtcatt gatcttggat gtggcagagg  7921 gggctggagt tactacgtcg ccaccatccg caaagttcaa gaagtgaaag gatacacaaa  7981 aggaggccct ggtcatgaag aacccgtgtt ggtgcaaagc tatgggtgga acatagtccg  8041 tcttaagagt ggggtggacg tctttcatat ggcggctgag ccgtgtgaca cgttgctgtg  8101 tgacataggt gagtcatcat ctagtcctga agtggaagaa gcacggacgc tcagagtcct  8161 ctccatggtg ggggattggc ttgaaaaaag accaggagcc ttttgtataa aagtgttgtg  8221 cccatacacc agcactatga tggaaaccct ggagcgactg cagcgtaggt atgggggagg  8281 actggtcaga gtgccactct cccgcaactc tacacatgag atgtactggg tctctggagc  8341 gaaaagcaac accataaaaa gtgtgtccac cacgagccag ctcctcttgg ggcgcatgga  8401 cgggcctagg aggccagtga aatatgagga ggatgtgaat ctcggctctg gcacgcgggc  8461 tgtggtaagc tgcgctgaag ctcccaacat gaagatcatt ggtaaccgca ttgaaaggat  8521 ccgcagtgag cacgcggaaa cgtggttctt tgacgagaac cacccatata ggacatgggc  8581 ttaccatgga agctatgagg cccccacaca agggtcagcg tcctctctaa taaacggggt  8641 tgtcaggctc ctgtcaaaac cctgggatgt ggtgactgga gtcacaggaa tagccatgac  8701 cgacaccaca ccgtatggtc agcaaagagt tttcaaggaa aaagtggaca ctagggtgcc  8761 agacccccaa gaaggcactc gtcaggttat gagcatggtc tcttcctggt tgtggaaaga  8821 gctaggcaaa cacaaacggc cacgagtctg caccaaagaa gagttcatca acaaggttcg  8881 tagcaatgca gcattagggg caatatttga agaggaaaaa gagtggaaga ctgcagtgga  8941 agctgtgaac gatccaaggt tctgggctct agtggacaag gaaagagagc accacctgag  9001 aggagagtgc cagagctgtg tgtacaacat gatgggaaaa agagaaaaga aacaagggga  9061 atttggaaag gccaagggca gccgcgccat ctggtatatg tggctagggg ctagatttct  9121 agagttcgaa gcccttggat tcttgaacga ggatcactgg atggggagag agaactcagg  9181 aggtggtgtt gaagggctgg gattacaaag actcggatat gtcctagaag agatgagtcg  9241 tataccagga ggaaggatgt atgcagatga cactgctggc tgggacaccc gcattagcag  9301 gtttgatctg gagaatgaag ctctaatcac caaccaaatg gagaaagggc acagggcctt  9361 ggcattggcc ataatcaagt acacatacca aaacaaagtg gtaaaggtcc ttagaccagc  9421 tgaaaaaggg aaaacagtta tggacattat ttcgagacaa gaccaaaggg ggagcggaca  9481 agttgtcact tacgctctta acacatttac caacctagtg gtgcaactca ttcggaatat  9541 ggaggctgag gaagttctag agatgcaaga cttgtggctg ctgcggaggt cagagaaagt  9601 gaccaactgg ttgcagagca acggatggga taggctcaaa cgaatggcag tcagtggaga  9661 tgattgcgtt gtgaagccaa ttgatgatag gtttgcacat gccctcaggt tcttgaatga  9721 tatgggaaaa gttaggaagg acacacaaga gtggaaaccc tcaactggat gggacaactg  9781 ggaagaagtt ccgttttgct cccaccactt caacaagctc catctcaagg acgggaggtc  9841 cattgtggtt ccctgccgcc accaagatga actgattggc cgggcccgcg tctctccagg  9901 ggcgggatgg agcatccggg agactgcttg cctagcaaaa tcatatgcgc aaatgtggca  9961 gctcctttat ttccacagaa gggacctccg actgatggcc aatgccattt gttcatctgt 10021 gccagttgac tgggttccaa ctgggagaac tacctggtca atccatggaa agggagaatg 10081 gatgaccact gaagacatgc ttgtggtgtg gaacagagtg tggattgagg agaacgacca 10141 catggaagac aagaccccag ttacgaaatg gacagacatt ccctatttgg gaaaaaggga 10201 agacttgtgg tgtggatctc tcatagggca cagaccgcgc accacctggg ctgagaacat 10261 taaaaacaca gtcaacatgg tgcgcaggat cataggtgat gaagaaaagt acatggacta 10321 cctatccacc caagttcgct acttgggtga agaagggtct acacctggag tgctgtaagc 10381 accaatctta atgttgtcag gcctgctagt cagccacagc ttggggaaag ctgtgcagcc 10441 tgtgaccccc ccaggagaag ctgggaaacc aagcctatag tcaggccgag aacgccatgg 10501 cacggaagaa gccatgctgc ctgtgagccc ctcagaggac actgagtcaa aaaaccccac 10561 gcgcttggag gcgcaggatg ggaaaagaag gtggcgacct tccccaccct tcaatctggg 10621 gcctgaactg gagatcagct gtggatctcc agaagaggga ctagtggtta gagga 

In some embodiments, the Zika virus may comprise the genome sequence ofGenBank Accession number KU501215.1. In some embodiments, the Zika virusis from strain PRVABC59. In some embodiments the genome sequence ofGenBank Accession number KU501215.1 comprises the sequence of SEQ ID NO:2. In some embodiments, the Zika virus may comprise a genomic sequencethat has at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity with the sequence of SEQ ID NO: 2.

In some embodiments, the Zika virus may comprise at least onepolypeptide encoded by the sequence of SEQ ID NO: 2. In someembodiments, the Zika virus may comprise at least one polypeptide havingan amino acid sequence that has at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% sequence identity with an aminoacid sequence encoded by the sequence of SEQ ID NO: 2.

Accordingly, in some embodiments, Zika viruses of the present disclosuremay be used in any of the vaccines and/or immunogenic compositionsdisclosed herein. For example, Zika viruses of the present disclosuremay be used to provide one or more antigens useful for treating orpreventing Zika virus infection in a human subject in need thereofand/or for inducing an immune response, such as a protective immuneresponse, against Zika virus in a human subject in need thereof.

Viral Antigens

In some embodiments, the present disclosure relates to one or moreantigens from any Zika virus described herein useful in vaccines and/orimmunogenic compositions including, without limitation, purifiedviruses, inactivated viruses, attenuated viruses, recombinant viruses,or purified and/or recombinant viral proteins for subunit vaccines. Insome embodiments, the vaccines and/or immunogenic compositions includeinactivated whole viruses.

Antigens of the present disclosure may be any substance capable ofeliciting an immune response. Examples of suitable antigens include, butare not limited to, whole virus, attenuated virus, inactivated virus,proteins, polypeptides (including active proteins and individualpolypeptide epitopes within proteins), glycopolypeptides,lipopolypeptides, peptides, polysaccharides, polysaccharide conjugates,peptide and non-peptide mimics of polysaccharides and other molecules,small molecules, lipids, glycolipids, and carbohydrates.

Antigens of the present disclosure may be from any Zika virus (e.g., aZika virus clonal isolate) produced from one or more cells in cellculture (e.g., via plaque purification). Any suitable cells known in theart for producing Zika virus may be used, including, for example, insectcells (e.g., mosquito cells such as CCL-125 cells, Aag-2 cells, RML-12cells, C6/36 cells, C7-10 cells, AP-61 cells, A.t. GRIP-1 cells, A.t.GRIP-2 cells, A.t. GRIP-3 cells, UM-AVE1 cells, Mos.55 cells, Sua1Bcells, 4a-3B cells, Mos.42 cells, MSQ43 cells, LSB-AA695BB cells,NIID-CTR cells, TRA-171, cells, and additional cells or cell lines frommosquito species such as Aedes aegypti, Aedes albopictus, Aedespseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae,Anopheles stephensi, Anopheles albimus, Culex quinquefasciatus, Culextheileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus, and/orToxorhynchites amboinensis), and mammalian cells (e.g., VERO cells (frommonkey kidneys), LLC-MK2 cells (from monkey kidneys), MDBK cells, MDCKcells, ATCC CCL34 MDCK (NBL2) cells, MDCK 33016 (deposit number DSM ACC2219 as described in WO97/37001) cells, BHK21-F cells, HKCC cells, orChinese hamster ovary cells (CHO cells). In some embodiments, antigensof the present disclosure are from a Zika virus (e.g., a Zika virusclonal isolate) produced from a non-human cell (e.g., via plaquepurification). In some embodiments, antigens of the present disclosureare from a Zika virus (e.g., a Zika virus clonal isolate) produced froman insect cell (e.g., via plaque purification). In some embodiments,antigens of the present disclosure are from a Zika virus (e.g., a Zikavirus clonal isolate) produced from a mosquito cell (e.g., via plaquepurification). In some embodiments, antigens of the present disclosureare from a Zika virus (e.g., a Zika virus clonal isolate) produced froma mammalian cell (e.g., via plaque purification). In some embodiments,antigens of the present disclosure are from a Zika virus (e.g., a Zikavirus clonal isolate) produced from a VERO cell (e.g., via plaquepurification). Methods of purifying a virus by performing plaquepurification are known to one of ordinary skill in the art (See e.g.,Example 1 below).

Antigens of the present disclosure may include at least one non-humancell adaptation mutation. Adaptation mutations may be generated byadapting a virus to growth in a particular cell line. For example, acell may be transfected or electroporated with a virus, RNA transcribedfrom a virus (e.g., an infectious virus, or infectious clone), and/orRNA purified from a whole virus and passaged such that the virus and/orviral RNA replicates and its nucleic acid mutates. Nucleic acidmutations may be point mutations, insertion mutations, or deletionmutations. Nucleic acid mutations may lead to amino acid changes withinviral proteins that facilitate growth of the virus in a non-human cell.Adaptation mutations may facilitate phenotypic changes in the virus,including altered plaque size, growth kinetics, temperature sensitivity,drug resistance, virulence, and virus yield in cell culture. Theseadaptive mutations may be useful in vaccine manufacture by increasingthe speed, yield, and consistency of virus cultured in a cell line.Adaptive mutations may change (e.g., enhance or decrease) immunogenicityof viral antigens by altering the structure of immunogenic epitopes. Inaddition, adaptive mutations may also increase the genetic stability ofthe virus and/or reduce or otherwise inhibit the development ofundesirable mutations in the virus through multiple (e.g., at leastfive, at least six, at least seven, at least eight, at least nine, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, at least 20, ormore) passages.

Accordingly, in certain embodiments, antigens of the present disclosureinclude at least one non-human cell adaptation mutation. In certainembodiments, the adaptation mutation is a mutation of a viral antigen toa non-human cell. In some embodiments, the non-human cell is a mammaliancell. Any suitable mammalian cell known in the art may be used,including, without limitation, VERO cells (from monkey kidneys), LLC-MK2cells (from monkey kidneys), MDBK cells, MDCK cells, ATCC CCL34 MDCK(NBL2) cells, MDCK 33016 (deposit number DSM ACC 2219 as described inWO97/37001) cells, BHK21-F cells, HKCC cells, or Chinese hamster ovarycells (CHO cells). In some embodiments, the non-human cell is a monkeycell. In some embodiments, the monkey cell is from a Vero cell line. Anysuitable Vero cell line known in the art may be used, including, withoutlimitation, WHO Vero 10-87, ATCC CCL-81, Vero 76 (ATCC Accession No.CRL-1587), or Vero C1008 (ATCC Accession No. CRL-1586). In someembodiments, the Vero cell line is WHO Vero 10-87.

Zika viruses possess a positive sense, single-stranded RNA genomeencoding both structural and nonstructural polypeptides. The genome alsocontains non-coding sequences at both the 5′- and 3′-terminal regionsthat play a role in virus replication. Structural polypeptides encodedby these viruses include, without limitation, capsid (C), precursormembrane (prM), and envelope (E). Non-structural (NS) polypeptidesencoded by these viruses include, without limitation, NS1, NS2A, NS2B,NS3, NS4A, NS4B, and NS5.

In certain embodiments, antigens of the present disclosure may containat least one (e.g., at least one, at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, at least 16, at least 17, at least 18, at least 19, atleast 20, etc.) non-human cell adaptation mutations within one or more(e.g., one or more, two or more, three or more, four or more, five ormore, six or more, seven or more, eight or more, nine or more, or allten) viral antigens/polypeptides, including, without limitation, C, prM,E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. In some embodiments,antigens of the present disclosure include at least one non-human celladaptation mutation in Zika virus Non-structural protein 1 (NS1). Insome embodiments, antigens of the present disclosure include whole,inactivated virus that may contain at least one (e.g., at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, at least 10, etc.)non-human cell adaptation mutations. In some embodiments, antigens ofthe present disclosure include whole, inactivated virus that may containat least one non-human cell adaptation mutation in Zika virusNon-structural protein 1 (NS1).

In some embodiments, the at least one non-human cell adaptation mutationis within the NS1 polypeptide. The amino acid sequence of a wild-type,non-cell adapted NS1 polypeptide from an exemplary Zika virus strain isset forth as:

(SEQ ID NO: 1) DVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWEDGICGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPQRLPVPVNELPHGWKAWGKSYFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSVWLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSPRAKDGCWYGMEIRPRKEPESNLVRSMV T. 

In some embodiments, the amino acid sequence of the NS1 polypeptide hasat least 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% sequence identity with the sequence of SEQ ID NO: 1. In someembodiments, the amino acid sequence of the NS1 polypeptide may be fromthe amino acid sequence encoded by the sequence of GenBank Accessionnumber KU501215.1 (SEQ ID NO: 2). In some embodiments, the amino acidsequence of the NS1 polypeptide may be amino acid positions 795 to 1145of the amino acid sequence encoded by the sequence of GenBank Accessionnumber KU501215.1. In some embodiments, the amino acid sequence of theNS1 polypeptide may be from Zika virus strain PRVABC59.

“Sequence Identity”, “% sequence identity”, “% identity”, “% identical”or “sequence alignment” means a comparison of a first amino acidsequence to a second amino acid sequence, or a comparison of a firstnucleic acid sequence to a second nucleic acid sequence and iscalculated as a percentage based on the comparison. The result of thiscalculation can be described as “percent identical” or “percent ID.”

Generally, a sequence alignment can be used to calculate the sequenceidentity by one of two different approaches. In the first approach, bothmismatches at a single position and gaps at a single position arecounted as non-identical positions in final sequence identitycalculation. In the second approach, mismatches at a single position arecounted as non-identical positions in final sequence identitycalculation; however, gaps at a single position are not counted(ignored) as non-identical positions in final sequence identitycalculation. In other words, in the second approach gaps are ignored infinal sequence identity calculation. The difference between these twoapproaches, i.e. counting gaps as non-identical positions vs ignoringgaps, at a single position can lead to variability in the sequenceidentity value between two sequences.

A sequence identity is determined by a program, which produces analignment, and calculates identity counting both mismatches at a singleposition and gaps at a single position as non-identical positions infinal sequence identity calculation. For example program Needle (EMBOS),which has implemented the algorithm of Needleman and Wunsch (Needlemanand Wunsch, 1970, J. Mol. Biol. 48: 443-453), and which calculatessequence identity per default settings by first producing an alignmentbetween a first sequence and a second sequence, then counting the numberof identical positions over the length of the alignment, then dividingthe number of identical residues by the length of an alignment, thenmultiplying this number by 100 to generate the % sequence identity [%sequence identity=(#of Identical residues/length of alignment)×100)].

A sequence identity can be calculated from a pairwise alignment showingboth sequences over the full length, so showing the first sequence andthe second sequence in their full length (“Global sequence identity”).For example, program Needle (EMBOSS) produces such alignments;% sequenceidentity=(#of identical residues/length of alignment)×100)].

A sequence identity can be calculated from a pairwise alignment showingonly a local region of the first sequence or the second sequence (“LocalIdentity”). For example, program Blast (NCBI) produces such alignments;%sequence identity=(#of Identical residues/length of alignment)×100)].

The sequence alignment is preferably generated by using the algorithm ofNeedleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453). Preferably,the program “NEEDLE” (The European Molecular Biology Open Software Suite(EMBOSS)) is used with the programs default parameter (gap open=10.0,gap extend=0.5 and matrix=EBLOSUM62 for proteins and matrix=EDNAFULL fornucleotides). Then, a sequence identity can be calculated from thealignment showing both sequences over the full length, so showing thefirst sequence and the second sequence in their full length (“Globalsequence identity”). For example:% sequence identity=(#of identicalresidues/length of alignment)×100)].

In some embodiments, the at least one non-human cell adaptation mutationoccurs at one or more amino acid positions within the NS1 polypeptide.In some embodiments, the mutation occurs at position 98 of SEQ ID NO: 1,or at a position corresponding to position 98 of SEQ ID NO: 1 whenaligned to SEQ ID NO: 1 using a pairwise alignment algorithm. In someembodiments, the mutation at position 98 is a tryptophan to glycinesubstitution.

In some embodiments, the Zika virus comprises a mutation at position 98of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQ IDNO: 1. A position corresponding to position 98 of SEQ ID NO:1 can bedetermined by aligning the amino acid sequence of an NS-1 protein to SEQID NO: 1 using a pairwise alignment algorithm. Amino acid residues inviruses other than Zika virus which correspond to the tryptophan residueat position 98 of SEQ ID NO:1 are shown in FIG. 7 of the presentapplication where these residues are boxed. In some embodiments, themutation at position 98 is a tryptophan to glycine substitution. In someembodiments, the mutation at position 98 is a tryptophan to glycinesubstitution at position 98 of SEQ ID NO:1.

In some embodiments, antigens of the present disclosure contain at leastone non-human cell adaptation mutation within the NS1 protein, andcontain at least one mutation (e.g., at least one adaptation mutation)within one or more of the C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B,and NS5 viral proteins. In some embodiments, antigens of the presentdisclosure contain one or more non-human cell adaptation mutationswithin the NS1 protein, and do not contain at least one mutation (e.g.,at least one non-human cell adaptation mutation) within one or more ofthe C, prM, E, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 viral proteins.In some embodiments, antigens of the present disclosure contain at leastone non-human cell adaptation mutation within the NS1 protein and do notcontain at least one mutation (e.g., at least one non-human celladaptation mutation) within the envelope protein E. In some embodiments,antigens of the present disclosure include whole, inactivated virus thatcontains at least one non-human cell adaptation mutation in Zika virusNon-structural protein 1 (NS1), and do not include a mutation in Zikavirus envelope protein E (Env). In some embodiments, antigens of thepresent disclosure contain a mutation at position 98 of SEQ ID NO: 1, orat a position corresponding to position 98 of SEQ ID NO: 1 and do notcontain any mutation within the envelope protein E. In some embodiments,antigens of the present disclosure include whole, inactivated virus thatcontains a mutation at position 98 of SEQ ID NO: 1, or at a positioncorresponding to position 98 of SEQ ID NO: 1 and do not include amutation in Zika virus envelope protein E (Env). In some embodiments,whole, inactivated virus contains at least one mutation in Zika virusNon-structural protein 1 (NS1) and the sequence encoding the envelopeprotein is the same as the corresponding sequence in SEQ ID No. 2. Insome embodiments, the Zika virus contains a mutation at position 98 ofSEQ ID NO: 1, or at a position corresponding to position 98 of SEQ IDNO: 1 and the sequence encoding the envelope protein is the same as thecorresponding sequence in SEQ ID No. 2. In some embodiments, whole,inactivated Zika virus contains a mutation at position 98 of SEQ ID NO:1, or at a position corresponding to position 98 of SEQ ID NO: 1 and thesequence encoding the envelope protein is the same as the correspondingsequence in SEQ ID No. 2.

In some embodiments, antigens of the present disclosure, such as Zikavirus, contain at least one non-human cell adaptation mutation thatenhances genetic stability as compared to a Zika virus lacking the atleast one adaptation mutation. In some embodiments, antigens of thepresent disclosure, such as Zika virus, contain at least one non-humancell adaptation mutation that enhances viral replication as compared toa Zika virus lacking the at least one adaptation mutation. In someembodiments, antigens of the present disclosure, such as Zika virus,contain at least one non-human cell adaptation mutation reduces orotherwise inhibits the occurrence of undesirable mutations, such aswithin the envelope protein E (Env) of the Zika virus.

In the above embodiments of the present disclosure, an exemplarypairwise alignment algorithm is the Needleman-Wunsch global alignmentalgorithm, using default parameters (e.g. with Gap opening penalty=10.0,and with Gap extension penalty=0.5, using the EBLOSUM62 scoring matrix).This algorithm is conveniently implemented in the needle tool in theEMBOSS package.

In some embodiments, antigens of the present disclosure from a Zikavirus may be used in any of the vaccines and immunogenic compositions ofthe present disclosure. For example, the antigens of the presentdisclosure may be useful for treating or preventing Zika virus infectionin a human subject in need thereof and/or inducing an immune response,such as a protective immune response, against Zika virus in a humansubject in need thereof.

Production of Vaccines and Immunogenic Compositions

Other aspects of the present disclosure relate to Zika virus vaccinesand immunogenic compositions containing one or more antigens of thepresent disclosure from at least one Zika virus. Such vaccines andimmunogenic compositions may be useful, for example, for treating orpreventing Zika virus infection in a human subject in need thereofand/or inducing an immune response, such as a protective immuneresponse, against Zika virus in a human subject in need thereof.Vaccines and/or immunogenic compositions of the present disclosure mayinclude, without limitation, purified viruses, inactivated viruses,attenuated viruses, recombinant viruses, purified and/or recombinantviral proteins for subunit vaccines. Vaccines and/or immunogeniccompositions of the present disclosure may further include a purifiedantigen vaccine or immunogenic composition, a subunit vaccine orimmunogenic composition, an inactivated whole virus vaccine orimmunogenic composition, or a purified inactivated whole virus vaccineor immunogenic composition or an attenuated virus vaccine or immunogeniccomposition.

Production of vaccines and/or immunogenic compositions of the presentdisclosure includes growth of Zika virus, with antigens being preparedfrom the grown virus. Growth in cell culture is a method for preparingvaccines and/or immunogenic compositions of the present disclosure.Cells for viral growth may be cultured in suspension or in adherentconditions.

Cell lines suitable for growth of the at least one virus of the presentdisclosure are preferably of mammalian origin, and include, but are notlimited to: insect cells (e.g., mosquito cells as described herein, VEROcells (from monkey kidneys), horse, cow (e.g. MDBK cells), sheep, dog(e.g. MDCK cells from dog kidneys, ATCC CCL34 MDCK (NBL2) or MDCK 33016,deposit number DSM ACC 2219 as described in WO97/37001), cat, and rodent(e.g. hamster cells such as BHK21-F, HKCC cells, or Chinese hamsterovary cells (CHO cells)), and may be obtained from a wide variety ofdevelopmental stages, including for example, adult, neonatal, fetal, andembryo. In certain embodiments, the cells are immortalized (e.g. PERC.6cells, as described in WO 01/38362 and WO 02/40665, and as depositedunder ECACC deposit number 96022940). In preferred embodiments,mammalian cells are utilized, and may be selected from and/or derivedfrom one or more of the following non-limiting cell types: fibroblastcells (e.g. dermal, lung), endothelial cells (e.g. aortic, coronary,pulmonary, vascular, dermal microvascular, umbilical), hepatocytes,keratinocytes, immune cells (e.g. T cell, B cell, macrophage, NK,dendritic), mammary cells (e.g. epithelial), smooth muscle cells (e.g.vascular, aortic, coronary, arterial, uterine, bronchial, cervical,retinal pericytes), melanocytes, neural cells (e.g. astrocytes),prostate cells (e.g. epithelial, smooth muscle), renal cells (e.g.epithelial, mesangial, proximal tubule), skeletal cells (e.g.chondrocyte, osteoclast, osteoblast), muscle cells (e.g. myoblast,skeletal, smooth, bronchial), liver cells, retinoblasts, and stromalcells. WO97/37000 and WO97/37001 describe production of animal cells andcell lines that are capable of growth in suspension and in serum freemedia and are useful in the production and replication of viruses.

Culture conditions for the above cell types are known and described in avariety of publications. Alternatively culture medium, supplements, andconditions may be purchased commercially, such as for example, describedin the catalog and additional literature of Cambrex Bioproducts (EastRutherford, N.J.).

In certain embodiments, the cells used in the methods described hereinare cultured in serum free and/or protein free media. A medium isreferred to as a serum-free medium in the context of the presentdisclosure in which there are no additives from serum of human or animalorigin. Protein-free is understood to mean cultures in whichmultiplication of the cells occurs with exclusion of proteins, growthfactors, other protein additives and non-serum proteins, but canoptionally include proteins such as trypsin or other proteases that maybe necessary for viral growth. The cells growing in such culturesnaturally contain proteins themselves.

Known serum-free media include Iscove's medium, Ultra-CHO medium(BioWhittaker) or EX-CELL (JRH Bioscience). Ordinary serum-containingmedia include Eagle's Basal Medium (BME) or Minimum Essential Medium(MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified EagleMedium (DMEM or EDM), which are ordinarily used with up to 10% fetalcalf serum or similar additives. Optionally, Minimum Essential Medium(MEM) (Eagle, Science, 130, 432 (1959)) or Dulbecco's Modified EagleMedium (DMEM or EDM) may be used without any serum containingsupplement. Protein-free media like PF-CHO (JHR Bioscience),chemically-defined media like ProCHO 4CDM (BioWhittaker) or SMIF 7(Gibco/BRL Life Technologies) and mitogenic peptides like Primactone,Pepticase or HyPep™ (all from Quest International) or lactalbuminhydrolysate (Gibco and other manufacturers) are also adequately known inthe prior art. The media additives based on plant hydrolysates have thespecial advantage that contamination with viruses, mycoplasma or unknowninfectious agents can be ruled out.

Cell culture conditions (temperature, cell density, pH value, etc.) arevariable over a very wide range owing to the suitability of the cellline employed according to the present disclosure and can be adapted tothe requirements of particular viral strains.

The method for propagating virus in cultured cells generally includesthe steps of inoculating the cultured cells with the strain to becultured, cultivating the infected cells for a desired time period forvirus propagation, such as for example as determined by virus titer orantigen expression (e.g. between 24 and 168 hours after inoculation) andcollecting the propagated virus. In some embodiments, the virus iscollected via plaque purification. The cultured cells are inoculatedwith a virus (measured by PFU or TCID50) to cell ratio of 1:500 to 1:1,preferably 1:100 to 1:5. The virus is added to a suspension of the cellsor is applied to a monolayer of the cells, and the virus is absorbed onthe cells for at least 10 minutes, at least 20 minutes, at least 30minutes, at least 40 minutes, at least 50 minutes, at least 60 minutesbut usually less than 300 minutes at 25° C. to 40° C., preferably 28° C.to 38° C. The infected cell culture (e.g. monolayers) may be removedeither by freeze-thawing or by enzymatic action to increase the viralcontent of the harvested culture supernatants. The harvested fluids arethen either inactivated or stored frozen. Cultured cells may be infectedat a multiplicity of infection (“MOI”) of about 0.0001 to 10, preferably0.002 to 5, more preferably to 0.001 to 2. Still more preferably, thecells are infected at an MOI of about 0.01. Infected cells may beharvested from 30 to 60 hours post infection, or 3 to 10 days postinfection. In certain preferred embodiments, the cells are harvested 3to 7 days post infection. More preferably, the cells are harvested 3 to5 days post infection. In some embodiments, proteases (e.g., trypsin)may be added during cell culture to allow viral release, and theproteases may be added at any suitable stage during the culture.Alternatively, in certain embodiments, the supernatant of infected cellcultures may be harvested and the virus may be isolated or otherwisepurified from the supernatant.

The viral inoculum and the viral culture are preferably free from (i.e.will have been tested for and given a negative result for contaminationby) herpes simplex virus, respiratory syncytial virus, parainfluenzavirus 3, SARS coronavirus, adenovirus, rhinovirus, reoviruses,polyomaviruses, birnaviruses, circoviruses, and/or parvoviruses[WO2006/027698].

Where virus has been grown on a cell line then it is standard practiceto minimize the amount of residual cell line DNA in the final vaccine,in order to minimize any oncogenic activity of the host cell DNA.Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination disclosed in references (Lundblad (2001) Biotechnology andApplied Biochemistry 34:195-197, Guidance for Industry: BioanalyticalMethod Validation. U.S. Department of Health and Human Services Food andDrug Administration Center for Drug Evaluation and Research (CDER)Center for Veterinary Medicine (CVM). May 2001.) involves a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Removal by β-propiolactonetreatment can also be used. In one embodiment, the contaminating DNA isremoved by benzonase treatment of the culture supernatant.

Production of Antigen

Antigens of the present disclosure for use in vaccines and/orimmunogenic compositions including, without limitation, purifiedviruses, inactivated viruses, inactivated whole viruses, attenuatedviruses, recombinant viruses, or purified and/or recombinant viralproteins for subunit vaccines to treat and/or prevent Zika virusinfection and/or induce an immune response, such as a protective immuneresponse, against Zika virus, may be produced and/or purified orotherwise isolated by any suitable method known in the art. Antigens ofthe present disclosure may include, without limitation, whole virus,attenuated virus, inactivated virus, inactivated whole viruses,proteins, polypeptides (including active proteins and individualpolypeptide epitopes within proteins), glycopolypeptides,lipopolypeptides, peptides, polysaccharides, polysaccharide conjugates,peptide and non-peptide mimics of polysaccharides and other molecules,small molecules, lipids, glycolipids, and carbohydrates produced,derived, purified, and/or otherwise isolated from a Zika virus. Forexample, suitable antigens may include, without limitation, structuralpolypeptides such as C, prM, and/or E, and non-structural polypeptides,such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, and/or NS5 from Zika virus.

In one embodiment, the antigen of the present disclosure is a purifiedinactivated whole Zika virus.

Antigen of the present disclosure may be synthesized chemically orenzymatically, produced recombinantly, isolated from a natural source,or a combination of the foregoing. In certain embodiments, antigens ofthe present disclosure are produced, purified, isolated, and/or derivedfrom at least one Zika virus of the present disclosure. Antigens of thepresent disclosure may be purified, partially purified, or a crudeextract. In some embodiments, antigens of the present disclosure areviruses, such as inactivated viruses, produced as described in the abovesection entitled “Production of Vaccines and Immunogenic Compositions.”

In certain embodiments, one or more antigens of the present disclosuremay be produced by culturing a non-human cell. Cell lines suitable forproduction of the one or more antigens of the present disclosure mayinclude insect cells (e.g., any of the mosquito cells described herein).Cell lines suitable for production of the one or more antigens of thepresent disclosure may also be cells of mammalian origin, and include,but are not limited to: VERO cells (from monkey kidneys), horse, cow(e.g. MDBK cells), sheep, dog (e.g. MDCK cells from dog kidneys, ATCCCCL34 MDCK (NBL2) or MDCK 33016, deposit number DSM ACC 2219 asdescribed in WO97/37001), cat, and rodent (e.g. hamster cells such asBHK21-F, HKCC cells, or Chinese hamster ovary cells (CHO cells)), andmay be obtained from a wide variety of developmental stages, includingfor example, adult, neonatal, fetal, and embryo. In certain embodiments,the cells are immortalized (e.g. PERC.6 cells, as described inWO01/38362 and WO02/40665, and as deposited under ECACC deposit number96022940). In preferred embodiments, mammalian cells are utilized, andmay be selected from and/or derived from one or more of the followingnon-limiting cell types: fibroblast cells (e.g. dermal, lung),endothelial cells (e.g. aortic, coronary, pulmonary, vascular, dermalmicrovascular, umbilical), hepatocytes, keratinocytes, immune cells(e.g. T cell, B cell, macrophage, NK, dendritic), mammary cells (e.g.epithelial), smooth muscle cells (e.g. vascular, aortic, coronary,arterial, uterine, bronchial, cervical, retinal pericytes), melanocytes,neural cells (e.g. astrocytes), prostate cells (e.g. epithelial, smoothmuscle), renal cells (e.g. epithelial, mesangial, proximal tubule),skeletal cells (e.g. chondrocyte, osteoclast, osteoblast), muscle cells(e.g. myoblast, skeletal, smooth, bronchial), liver cells, retinoblasts,and stromal cells. WO97/37000 and WO97/37001 describe production ofanimal cells and cell lines that capable of growth in suspension and inserum free media and are useful in the production of viral antigens. Incertain embodiments, the non-human cell is cultured in serum-free media.

Polypeptide antigens may be isolated from natural sources using standardmethods of protein purification known in the art, including, but notlimited to, liquid chromatography (e.g., high performance liquidchromatography, fast protein liquid chromatography, etc.), sizeexclusion chromatography, gel electrophoresis (including one-dimensionalgel electrophoresis, two-dimensional gel electrophoresis), affinitychromatography, or other purification technique. In many embodiments,the antigen is a purified antigen, e.g., from about 50% to about 75%pure, from about 75% to about 85% pure, from about 85% to about 90%pure, from about 90% to about 95% pure, from about 95% to about 98%pure, from about 98% to about 99% pure, or greater than 99% pure. Thepurity of the purified antigen can be determined by size exclusionchromatography and the %-purity corresponds to the % of the main peak tothe total area under the curve. The main peak of the purified antigen inthe size exclusion chromatography may be more than 85% of the total areaunder the curve in the size exclusion chromatography, or more than 90%of the total area under the curve in the size exclusion chromatography,or more than 95%, or more than 98% or more than 99% of the total areaunder the curve in the size exclusion chromatography. Such results areconsidered as “purified” antigen within the meaning of this invention.

In accordance with the above disclosure regarding purity, the term“purified Zika virus” means that the main peak of the purified Zikavirus in the size exclusion chromatography is more than 85% of the totalarea under the curve in the size exclusion chromatography, or more than90% of the total area under the curve in the size exclusionchromatography, or more than 95%, more than 98% or more than 99% of thetotal area under the curve in the size exclusion chromatography.

In accordance with the above disclosure regarding purity, the term“purified inactivated whole Zika virus” means that the main peak of thepurified inactivated whole Zika virus in the size exclusionchromatography is more than 85% of the total area under the curve in thesize exclusion chromatography, or more than 90% of the total area underthe curve in the size exclusion chromatography, or more than 95%, morethan 98% or more than 99% of the total area under the curve in the sizeexclusion chromatography.

One may employ solid phase peptide synthesis techniques, where suchtechniques are known to those of skill in the art. See Jones, TheChemical Synthesis of Peptides (Clarendon Press, Oxford) (1994).Generally, in such methods a peptide is produced through the sequentialaddition of activated monomeric units to a solid phase bound growingpeptide chain.

Well-established recombinant DNA techniques can be employed forproduction of polypeptides, where, e.g., an expression constructcomprising a nucleotide sequence encoding a polypeptide is introducedinto an appropriate host cell (e.g., a eukaryotic host cell grown as aunicellular entity in in vitro cell culture, e.g., a yeast cell, aninsect cell, a mammalian cell, etc.) or a prokaryotic cell (e.g., grownin in vitro cell culture), generating a genetically modified host cell;under appropriate culture conditions, the protein is produced by thegenetically modified host cell.

Besides killed and attenuated virus immunogenic compositions, one canuse a subunit immunogenic composition or other type of immunogeniccomposition which presents to the animal the antigenic components ofZika virus. The antigenic component may be a protein, glycoprotein,lipid-conjugated protein or glycoprotein, a modified lipid moiety, orother viral component which, when injected into a human, stimulates animmune response in the human such that the human develops protectiveimmunity against Zika virus. For a subunit immunogenic composition, thevirus can be cultured on mammalian cells, as described above. The cellculture can be homogenized and an immunogenic composition can beisolated by passage of the cell culture homogenate over the appropriatecolumn or through the appropriate pore size filter or via centrifugationof the cell culture homogenate.

If the antigenic component is a protein, then one can isolate thenucleic acid which encodes that protein and generate an immunogeniccomposition that contains that isolated nucleic acid. The nucleic acidencoding the antigenic component can be placed on a plasmid downstreamof a signal sequence of a eukaryotic promoter. That plasmid can containone or more selectable markers and be transfected into an attenuatedprokaryotic organism, such as Salmonella spp., Shigella spp., or othersuitable bacteria. The bacteria can then be administered to the human sothat the human can generate a protective immune response to theantigenic component. Alternatively, the nucleic acid encoding theantigenic component can be placed downstream of a prokaryotic promoter,have one or more selectable markers, and be transfected into anattenuated prokaryotic organism such as Salmonella spp., Shigella spp.,or other suitable bacteria. The bacteria can then be administered to theeukaryotic human subject for which immune response to the antigen ofinterest is desired. See, for example, U.S. Pat. No. 6,500,419.

For a subunit immunogenic composition, the nucleic acid encoding aproteinaceous antigenic component of a Zika virus can be cloned into aplasmid such as those described in International Patent ApplicationPublication Number WO 00/32047 (Galen) and International PatentApplication Publication Number WO 02/083890 (Galen). Then the plasmidcan be transfected into bacteria and the bacteria can produce thedesired antigenic protein. One can isolate and purify the desiredantigenic protein by a variety of methods described in both patentapplications.

Virus Inactivation

Certain aspects of the present disclosure relate to Zika virus vaccinesand immunogenic compositions containing one or more antigens from a Zikavirus. Vaccines and/or immunogenic compositions of the presentdisclosure may include a purified virus, a whole virus, a recombinantvirus, a live attenuated whole virus or, preferably, an inactivatedwhole virus, or subunits, polypeptides, and/or antigens from aninactivated virus. As such, certain embodiments of the presentdisclosure relate to Zika virus vaccines and/or immunogenic compositionscontaining one or more antigens from at least one inactivated Zikavirus.

Methods of inactivating or killing viruses to destroy their ability toinfect mammalian cells but do not destroy the structure of the virus areknown in the art. Such methods include both chemical and physical means.Suitable means for inactivating a virus include, without limitation,treatment with an effective amount of one or more agents selected fromdetergents, formalin (also referred to herein as “formaldehyde”),beta-propiolactone (BPL), binary ethylamine (BEI), acetyl ethyleneimine,heat, electromagnetic radiation, x-ray radiation, gamma radiation,ultraviolet radiation (UV radiation), UV-A radiation, UV-B radiation,UV-C radiation, methylene blue, psoralen, carboxyfullerene (C60) and anycombination of any thereof. When reference is made herein to aconcentration of formaldehyde, it refers to the concentration offormaldehyde (and not to the concentration of formalin). Accordingly, a“formaldehyde concentration of 0.01% (w/v)” refers to 0.01% (w/v)formaldehyde, and no further correction of this concentration for theformaldehyde concentration in the formalin stock solution (whichtypically contains 37% formaldehyde by mass) has to be made. Forexample, such a formaldehyde concentration in the virus preparation canbe obtained by diluting formalin to a working solution having aformaldehyde content of 1.85% (w/v) which is then further diluted to therequired concentration when it is mixed with the virus preparation suchas the Zika virus preparation.

In certain embodiments of the present disclosure the at least one virusis chemically inactivated. Agents for chemical inactivation and methodsof chemical inactivation are well-known in the art and described herein.In some embodiments, the at least one virus is chemically inactivatedwith one or more of BPL, formalin, or BEI. In certain embodiments wherethe at least one virus is chemically inactivated with BPL, the virus maycontain one or more modifications. In some embodiments, the one or moremodifications may include a modified nucleic acid. In some embodiments,the modified nucleic acid is an alkylated nucleic acid. In otherembodiments, the one or more modifications may include a modifiedpolypeptide. In some embodiments, the modified polypeptide contains amodified amino acid residue including one or more of a modifiedcysteine, methionine, histidine, aspartic acid, glutamic acid, tyrosine,lysine, serine, and threonine.

In certain embodiments where the at least one virus is chemicallyinactivated with formalin, the inactivated virus may contain one or moremodifications. In some embodiments, the one or more modifications mayinclude a modified polypeptide. In some embodiments, the one or moremodifications may include a cross-linked polypeptide. In someembodiments where the at least one virus is chemically inactivated withformalin, the vaccine or immunogenic composition further includesformalin. In certain embodiments where the at least one virus ischemically inactivated with BEI, the virus may contain one or moremodifications. In some embodiments, the one or more modifications mayinclude a modified nucleic acid. In some embodiments, the modifiednucleic acid is an alkylated nucleic acid.

In some embodiments where the at least one virus is chemicallyinactivated with formalin, any residual unreacted formalin may beneutralized with sodium metabisulfite, may be dialyzed out, and/or maybe buffer exchanged to remove the residual unreacted formalin. In someembodiments, the sodium metabisulfite is added in excess. In someembodiments, the solutions may be mixed using a mixer, such as anin-line static mixer, and subsequently filtered or further purified(e.g., using a cross flow filtrations system).

Certain embodiments of the present disclosure relate to a method forinactivating a Zika virus preparation. In some embodiments, the methodinvolves (a) isolating, followed by purification the Zika viruspreparation from one or more non-human cells that are used to producethe virus preparation and (b) treating the virus preparation with aneffective amount of formalin.

Certain embodiments of the present disclosure relate to a method forinactivating a Zika virus preparation. In some embodiments, the methodcomprises:

(a) isolating the Zika virus preparation from one or more cells culturedin vitro, wherein the cells are used to produce the Zika viruspreparation, wherein isolating the Zika virus preparation comprises oneor more steps selected from: (i) depth filtration, (ii) buffer exchangeand/or dilution; (iii) ion exchange chromatography; and(b) treating the Zika virus preparation with formaldehyde, wherein thenumerical result of the multiplication of the formaldehyde concentrationas measured in % (w/v) with the period of incubation with formaldehydeas measured in days is 0.025 to 0.5.

In certain embodiments, treating with an effective amount of formalinincludes, without limitation, treating with formalin in an amount thatranges from about 0.001% v/v to about 3.0% v/v. For example, treatingwith an effective amount of formalin may include treating with formalinin an amount that ranges from about 0.001% to about 3.0% v/v, about0.005% to about 2.0% v/v, or about 0.01% to about 1.0% v/v, or in anamount of about 0.001%, about 0.0025%, about 0.005%, about 0.0075%,about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%,about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%,about 0.9%, about 1.0%, about 1.25%, about 1.5%, about 1.75%, about2.0%, about 2.25%, about 2.5%, about 2.75%, or about 3.0% v/v.

In certain embodiments of the method, the Zika virus preparation istreated with formalin at a temperature that ranges from about 2° C. toabout 42° C. For example, the Zika virus preparation may be treated withformalin at a temperature that ranges from about 2° C. to about 42° C.,about 2° C. to about 8° C., about 15° C. to about 37° C. about 17° C. toabout 27° C., about 20° C. to about 25° C., or at a temperature of about2° C., about 4° C., about 8° C., about 10° C., about 15° C., about 17°C., about 18° C., about 19° C., about 20° C., about 21° C., about 22°C., about 23° C., about 24° C., about 25° C., about 26° C., about 27°C., about 28° C., about 29° C., about 30° C., about 37° C., or about 42°C. In some embodiments, the Zika virus preparation is treated withformalin at room temperature.

In some embodiments, the Zika virus preparation is treated with formalinfor at least about 1 day. For example, the Zika virus preparation may betreated with formalin for at least about 1 day, at least about 2 days,at least about 3 days, at least about 4 days, at least about 5 days, atleast about 6 days, and e.g. for no more than 15 days, e.g. from 5 to 15days. For example, the Zika virus preparation may be treated withformalin for at least about 7 days, at least about 8 days, at leastabout 9 days, at least about 10 days, at least about 11 days, at leastabout 12 days, at least about 13 days, at least about 14 days, at leastabout 15 days, at least about 16 days, at least about 17 days, at leastabout 18 days, at least about 19 days, at least about 20 days, at leastabout 21 days, at least about 22 days, at least about 23 days, at leastabout 24 days, at least about 25 days, at least about 26 days, at leastabout 27 days, at least about 28 days, at least about 29 days, at leastabout 30 days, or more. In some embodiments, the Zika virus preparationis treated with formalin for at least about 9 days. In some embodiments,the Zika virus preparation is treated with formalin for at least about11 days. In some embodiments, the Zika virus preparation is treated withformalin for at least about 14 days. In some embodiments, the Zika viruspreparation is treated with formalin for at least about 20 days. In someembodiments, the Zika virus preparation is treated with formalin for atleast about 30 days.

In some embodiments, the Zika virus preparation is treated with 0.005 to0.02% (w/v) formalin for eight to twelve days at a temperature of 15° C.to 30° C. In some embodiments, the Zika virus preparation is treatedwith 0.005 to 0.02% (w/v) formalin for nine to eleven days at atemperature of 15° C. to 30° C. In some embodiments, the Zika viruspreparation is treated with 0.005 to 0.02% (w/v) formalin for ten daysat a temperature of 15° C. to 30° C. In some embodiments, the Zika viruspreparation is treated with 0.008 to 0.015% (w/v) formalin for eight totwelve days at a temperature of 15° C. to 30° C. In some embodiments,the Zika virus preparation is treated with 0.008 to 0.015% (w/v)formalin for nine to eleven days at a temperature of 15° C. to 30° C. Insome embodiments, the Zika virus preparation is treated with 0.008 to0.015% (w/v) formalin for ten days at a temperature of 15° C. to 30° C.In some embodiments, the Zika virus preparation is treated with 0.01%(w/v) formalin for eight to twelve days at a temperature of 15° C. to30° C. In some embodiments, the Zika virus preparation is treated with0.01% (w/v) formalin for nine to eleven days at a temperature of 15° C.to 30° C. In some embodiments, the Zika virus preparation is treatedwith 0.01% (w/v) formalin for ten days at a temperature of 15° C. to 30°C.

In some embodiments, the Zika virus preparation is treated with 0.005 to0.02% (w/v) formalin for eight to twelve days at a temperature of 18° C.to 25° C. In some embodiments, the Zika virus preparation is treatedwith 0.005 to 0.02% (w/v) formalin for nine to eleven days at atemperature of 18° C. to 25° C. In some embodiments, the Zika viruspreparation is treated with 0.005 to 0.02% (w/v) formalin for ten daysat a temperature of 18° C. to 25° C. In some embodiments, the Zika viruspreparation is treated with 0.008 to 0.015% (w/v) formalin for eight totwelve days at a temperature of 18° C. to 25° C. In some embodiments,the Zika virus preparation is treated with 0.008 to 0.015% (w/v)formalin for nine to eleven days at a temperature of 18° C. to 25° C. Insome embodiments, the Zika virus preparation is treated with 0.008 to0.015% (w/v) formalin for ten days at a temperature of 18° C. to 25° C.In some embodiments, the Zika virus preparation is treated with 0.01%(w/v) formalin for eight to twelve days at a temperature of 18° C. to25° C. In some embodiments, the Zika virus preparation is treated with0.01% (w/v) formalin for nine to eleven days at a temperature of 18° C.to 25° C. In some embodiments, the Zika virus preparation is treatedwith 0.01% (w/v) formalin for ten days at a temperature of 18° C. to 25°C.

In some embodiments, the Zika virus preparation is treated with 0.005 to0.02% (w/v) formalin for eight to twelve days at a temperature of 22° C.In some embodiments, the Zika virus preparation is treated with 0.005 to0.02% (w/v) formalin for nine to eleven days at a temperature of 22° C.In some embodiments, the Zika virus preparation is treated with 0.005 to0.02% (w/v) formalin for ten days at a temperature of 22° C. In someembodiments, the Zika virus preparation is treated with 0.008 to 0.015%(w/v) formalin for eight to twelve days at a temperature of 22° C. Insome embodiments, the Zika virus preparation is treated with 0.008 to0.015% (w/v) formalin for nine to eleven days at a temperature of 22° C.In some embodiments, the Zika virus preparation is treated with 0.008 to0.015% (w/v) formalin for ten days at a temperature of 22° C. In someembodiments, the Zika virus preparation is treated with 0.01% (w/v)formalin for eight to twelve days at a temperature of 22° C. In someembodiments, the Zika virus preparation is treated with 0.01% (w/v)formalin for nine to eleven days at a temperature of 22° C. In someembodiments, the Zika virus preparation is treated with 0.01% (w/v)formalin for ten days at a temperature of 22° C.

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.05 to 0.25. In some embodiments, the numerical resultof the multiplication of the formaldehyde concentration as measured in %(w/v) with the period of incubation with formaldehyde as measured indays is 0.075 to 0.15. In some embodiments, the numerical result of themultiplication of the formaldehyde concentration with the period ofincubation with formaldehyde is 0.1.

In some embodiments, the formaldehyde concentration is 0.005% (w/v) to0.02% (w/v). In some embodiments, the formaldehyde concentration is0.0075% (w/v) to 0.015% (w/v). In some embodiments, the formaldehydeconcentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration as measured in % (w/v) with the period ofincubation with formaldehyde as measured in days is 0.025 to 0.5 and theformaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In someembodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.025 to 0.5 and the formaldehyde concentration is0.0075% (w/v) to 0.015% (w/v). In some embodiments, the numerical resultof the multiplication of the formaldehyde concentration with the periodof incubation with formaldehyde is 0.025 to 0.5 and the formaldehydeconcentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration as measured in % (w/v) with the period ofincubation with formaldehyde as measured in days is 0.05 to 0.25 and theformaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In someembodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.05 to 0.25 and the formaldehyde concentration is0.0075% (w/v) to 0.015% (w/v). In some embodiments, the numerical resultof the multiplication of the formaldehyde concentration with the periodof incubation with formaldehyde is 0.05 to 0.25 and the formaldehydeconcentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration as measured in % (w/v) with the period ofincubation with formaldehyde as measured in days is 0.075 to 0.15 andthe formaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In someembodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.075 to 0.15 and the formaldehyde concentration is0.0075% (w/v) to 0.015% (w/v). In some embodiments, the numerical resultof the multiplication of the formaldehyde concentration with the periodof incubation with formaldehyde is 0.075 to 0.15 and the formaldehydeconcentration is 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration as measured in % (w/v) with the period ofincubation with formaldehyde as measured in days is 0.1 and theformaldehyde concentration is 0.005% (w/v) to 0.02% (w/v). In someembodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.1 and the formaldehyde concentration is 0.0075% (w/v)to 0.015% (w/v). In some embodiments, the numerical result of themultiplication of the formaldehyde concentration with the period ofincubation with formaldehyde is 0.1 and the formaldehyde concentrationis 0.01% (w/v).

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration as measured in % (w/v) with the period ofincubation with formaldehyde as measured in days is 0.025 to 0.5 and theperiod of incubation with formaldehyde is eight to twelve days. In someembodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.025 to 0.5 and the period of incubation withformaldehyde is nine to eleven days. In some embodiments, the numericalresult of the multiplication of the formaldehyde concentration with theperiod of incubation with formaldehyde is 0.025 to 0.5 and the period ofincubation with formaldehyde is ten days.

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration as measured in % (w/v) with the period ofincubation with formaldehyde as measured in days is 0.05 to 0.25 and theperiod of incubation with formaldehyde is eight to twelve days. In someembodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.05 to 0.25 and the period of incubation withformaldehyde is nine to eleven days. In some embodiments, the numericalresult of the multiplication of the formaldehyde concentration with theperiod of incubation with formaldehyde is 0.05 to 0.25 and the period ofincubation with formaldehyde is ten days.

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration as measured in % (w/v) with the period ofincubation with formaldehyde as measured in days is 0.075 to 0.15 andthe period of incubation with formaldehyde is eight to twelve days. Insome embodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.075 to 0.15 and the period of incubation withformaldehyde is nine to eleven days. In some embodiments, the numericalresult of the multiplication of the formaldehyde concentration with theperiod of incubation with formaldehyde is 0.075 to 0.15 and the periodof incubation with formaldehyde is ten days.

In some embodiments, the numerical result of the multiplication of theformaldehyde concentration as measured in % (w/v) with the period ofincubation with formaldehyde as measured in days is 0.1 and the periodof incubation with formaldehyde is eight to twelve days. In someembodiments, the numerical result of the multiplication of theformaldehyde concentration with the period of incubation withformaldehyde is 0.1 and the period of incubation with formaldehyde isnine to eleven days. In some embodiments, the numerical result of themultiplication of the formaldehyde concentration with the period ofincubation with formaldehyde is 0.1 and the period of incubation withformaldehyde is ten days.

In some embodiments, the method further involves neutralizing unreactedformalin with an effective amount of sodium metabisulfite. In someembodiments, the effective amount of sodium metabisulfite ranges fromabout 0.01 mM to about 100 mM. For example, the sodium metabisulfite maybe added at an effective concentration of from about 0.01 mM to about100 mM, from about 0.1 mM to about 50 mM, from about 0.5 mM to about 20mM, or from about 1 mM to about 10 mM, or at a concentration of about0.01 mM, about 0.05 mM, about 0.1 mM, about 0.25 mM, about 0.5 mM, about0.75 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM,about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20mM, about 30 mM about 40 mM, about 50 mM, about 75 mM or about 100 mM.In some embodiments, the formalin is neutralized with about 2 mM sodiummetabisulfite.

In some embodiments, the method involves (a) isolating followed bypurification of the Zika virus preparation from one or more non-humancells that are used to produce the virus preparation; (b) treating thevirus preparation with an effective amount of formalin; (c) neutralizingthe virus preparation with an effective amount of sodium metabisulfite;and (d) purifying the neutralized virus preparation. Any method ofpurifying a virus preparation known in the art may be employed,including, without limitation, using cross flow filtration (CFF),multimodal chromatography, size exclusion chromatography, cationexchange chromatography, and/or anion exchange chromatography. In someembodiments, the neutralized virus preparation is purified by cross flowfiltration (CFF). In some embodiments, the virus preparation is purifiedto a high degree in an amount that is about 70%, about 75%, about 80%,about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about95% about 96%, about 97%, about 98%, about 99%, or more.

Certain embodiments of the present disclosure relate thus to Zika virusvaccines and/or immunogenic compositions containing a purifiedinactivated whole Zika virus. The term “inactivated whole Zika virus” asused herein is intended to comprise a Zika virus, which has been treatedwith an inactivating method such as treatment with an effective amountof formalin. Such a treatment is considered not to destroy the structureof the virus, i.e. it does not destroy the secondary, tertiary orquaternary structure and immunogenic epitopes of the virus, but theinactivated Zika virus is no longer able to infect host cells, which canbe infected with a Zika virus that has not been inactivated. In oneembodiment, the inactivated Zika virus is no longer able to infect VEROcells and exert a cytopathic effect on the VERO cells.

The method for determining the completeness of inactivation of an Zikavirus preparation comprises the steps of:

-   -   (i) inoculating insect cells with an Zika virus preparation        which was subjected to an inactivation step and incubating the        insect cells for a first period of time, thereby producing an        insect cell supernatant;    -   (ii) inoculating mammalian cells with the insect cell        supernatant produced in (i) and incubating the mammalian cells        for a second period of time; and    -   (iii) determining whether the virus preparation contains a        residual replicating virus that produces a cytopathic effect on        the mammalian cells.

In some embodiments, the method for determining the completeness ofinactivation of an Zika virus preparation comprises the following steps:

-   -   (i) inoculating C6/36 cells with a Zika virus preparation which        was subjected to an inactivation step and incubating the C6/36        cells for a first period of time, thereby producing an C6/36        cell supernatant;    -   (ii) inoculating Vero cells with the C6/36 cell supernatant        produced in (i) and incubating the Vero cells for a second        period of time; and    -   (iii) determining whether the virus preparation contains a        residual replicating virus that produces a cytopathic effect on        the Vero cells.

At the end of the second period of time it is determined whether thevirus preparation has a cytopathic effect on the mammalian cells. Acytopathic effect is any change in the cell structure caused by viralinvasion, infection, and budding from the cells during viralreplication. In the method of the present disclosure, the cytopathiceffect is determined by a change in the media color from pink to orangeor yellow, if the cells are cultured in a medium containing phenol red,or by a microscopic examination of the mammalian cells. If themicroscopic examination of the mammalian cells shows that the cellsround, begin to pull away from the tissue culture vessel (plate, well orflask), or clear from the tissue culture plate/flask, it is consideredthat a cytopathic effect is present. Other indicia of a cytopathiceffect include the fusion of adjacent cells to form syncytia and theappearance of nuclear or cytoplasmic inclusion bodies.

As discussed above, the method disclosed herein has a very low limit ofdetection. With this method a virus content of less than 1.0 TCID50 canbe detected. In some embodiments, a virus content of less than 0.8TCID50 can be detected. In some embodiments, a virus content of lessthan 0.5 TCID50 can be detected. In some embodiments, a virus content ofless than 0.2 TCID50 can be detected. In some embodiments, a viruscontent of less than 0.1 TCID50 can be detected.

For the present disclosure the term “inactivated whole Zika virus” thusrefers in particular to a Zika virus obtainable from a method whereinthe Zika virus is treated with formalin in an amount that ranges fromabout 0.001% v/v to about 3.0% v/v from 5 to 15 days at a temperaturethat ranges from about 15° C. to about 37° C., in particular 0.02% v/vformaldehyde for 10 days at 22° C. or in particular 0.01% v/vformaldehyde for 10 days at 22° C. The definition is meant to encompassZika virus obtained from a method wherein the Zika virus is treated withformalin in an amount that ranges from about 0.001% v/v to about 3.0%v/v from 5 to 15 days at a temperature that ranges from about 15° C. toabout 37° C., in particular 0.02% v/v formaldehyde for 10 days at 22° C.or in particular 0.01% v/v formaldehyde for 10 days at 22° C., but isnot to be understood to be limited to those, since other methods maylead to the same inactivated whole Zika virus. In certain suchembodiments, however, the Zika virus is obtained from a method whereinthe Zika virus is treated with formalin in an amount that ranges fromabout 0.001% v/v to about 3.0% v/v from 5 to 15 days at a temperaturethat ranges from about 15° C. to about 37° C., in particular 0.02% v/vformaldehyde for 10 days at 22° C. or in particular 0.01% v/vformaldehyde for 10 days at 22° C.

Alternatively, within the present disclosure “inactivated whole Zikavirus” thus refers to a Zika virus that has been tested by the methodcomprising the steps (i) to (iii) and does not show any plaque formationin step (iii):

-   -   (i) inoculating insect cells with an Zika virus preparation        which was subjected to an inactivation step and incubating the        insect cells for a first period of time, thereby producing an        insect cell supernatant;    -   (ii) inoculating mammalian cells with the insect cell        supernatant produced in (i) and incubating the mammalian cells        for a second period of time; and    -   (iii) determining whether the virus preparation contains a        residual replicating virus that produces a cytopathic effect on        the mammalian cells.

The term “purified inactivated whole Zika virus” thus refers to a Zikavirus obtainable or obtained from a method wherein the Zika virus istreated with formalin in an amount that ranges from about 0.001% v/v toabout 3.0% v/v from 5 to 15 days at a temperature that ranges from about15° C. to about 37° C., in particular 0.02% v/v formaldehyde for 10 daysat 22° C. or in particular 0.01% v/v formaldehyde for 10 days at 22° C.,or alternatively by the above mentioned method for determining thecompleteness of inactivation and, if required, has been subjected to apurification process. The purified Zika virus has therefore a lowercontent of host cell proteins such as Vero cell proteins and host cellDNA such as Vero cell DNA than a non-purified Zika virus. The term“purified inactivated whole Zika virus” thus refers to a Zika virusobtainable or obtained from a method wherein the Zika virus is treatedwith formalin in an amount that ranges from about 0.001% v/v to about3.0% v/v from 5 to 15 days at a temperature that ranges from about 15°C. to about 37° C., in particular 0.02% v/v formaldehyde for 10 days at22° C. or in particular 0.01% v/v formaldehyde for 10 days at 22° C., oralternatively by the above mentioned method for determining thecompleteness of inactivation and, provides a main peak of at least 85%of the total area under the curve in the size exclusion chromatography.

In certain such embodiments the purified inactivated whole Zika virus isfurthermore a clonal isolate obtained or obtainable by plaquepurification.

In certain such embodiments the purified inactivated whole Zika virus,which is optionally furthermore a clonal isolate obtained or obtainableby plaque purification, contains a mutation at position 98 of SEQ ID NO:1, or at a position corresponding to position 98 of SEQ ID NO: 1 anddoes not contain any mutation within the envelope protein E. In certainsuch embodiments the mutation is a Trp98Gly mutation at position 98 ofSEQ ID NO: 1, or at a position corresponding to position 98 of SEQ IDNO:1. In certain such embodiments the Zika virus is derived from strainPRVABC59. In certain such embodiments the Zika virus is derived fromstrain PRVABC59 comprising the genomic sequence according to SEQ IDNO:2.

The vaccines and/or immunogenic compositions of the present disclosurecontaining one or more antigens from at least one inactivated Zika virusmay be useful for treating or preventing Zika virus infection in a humansubject in need thereof and/or inducing an immune response, such as aprotective immune response, against Zika virus in a human subject inneed thereof.

Other aspects of the present disclosure relate to vaccine or immunogeniccomposition comprising an inactivated Zika virus which is obtainable bythe methods disclosed herein. These vaccines or immunogenic compositionshave a particularly low content of residual formaldehyde.

The term “residual formaldehyde content” refers to the amount offormaldehyde which is still present in the vaccine or immunogeniccomposition after the Zika virus has been inactivated and thepreparation has been neutralized and optionally subjected to one or morefurther purification or filtration steps. According to the USpharmacopoeia the upper limit for residual formaldehyde in vaccinescomprising inactivated bacteria or viruses is 0.02% which is equivalentto 100 μg/ml formaldehyde.

The residual formaldehyde content can be determined by any method knownto the skilled person. One suitable method is described in EMEA, VICHTopic GL25, Biologicals: Testing of residual formaldehyde, 30 Apr. 2002and involves the use of Methylbenzothiazolone hydrazone hydrochloride(MBTH). Other methods include acetyl acetone titration, ferric chloridetitration and the basic fuchsin test. A particularly suitable method isdescribed herein.

The vaccine or immunogenic composition is in a form which can beadministered to a subject and typically contains one or morepharmaceutically acceptable excipients.

The content of residual formaldehyde in the vaccine or immunogeniccomposition is less than 50 μg/ml. In one embodiment, the residualformaldehyde content in the vaccine or immunogenic composition is lessthan 45 μg/ml, less than 40 μg/ml, less than 35 μg/ml, less than 30μg/ml, less than 25 μg/ml, less than 20 μg/ml, less than 15 μg/ml orless than 10 μg/ml. In one embodiment, the residual formaldehyde contentin the vaccine or immunogenic composition is less than 9.5 μg/ml, lessthan 9 μg/ml, less than 8.5 μg/ml, less than 8 μg/ml, less than 7.5μg/ml, less than 7 μg/ml, less than 6.5 μg/ml, less than 6 μg/ml, lessthan 5.5 μg/ml, less than 5 μg/ml, less than 4.5 μg/ml, less than 4μg/ml, less than 3.5 μg/ml, less than 3 μg/ml, less than 2.5 μg/ml, lessthan 2 μg/ml, less than 1.5 μg/ml, less than 1 μg/ml or less than 0.5μg/ml. In one embodiment, the residual formaldehyde content in thevaccine or immunogenic composition is less than 0.5 μg/ml.

Methods for Determining Residual Formaldehyde Content

Other aspects of the present disclosure relate to a method fordetermining the residual formaldehyde content in a vaccine orimmunogenic composition comprising an inactivated virus, comprising thesteps of:

(a) providing a composition comprising a virus which has been treatedwith formaldehyde;(b) mixing the composition of (a) with phosphoric acid and2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;(c) incubating the mixture of (b) under suitable conditions; and(d) analyzing the mixture for the presence of residual formaldehyde.

The use of DNPH as detection reagent offers the following advantages:(1) high sensitivity, (2) UV detection of the derivatized formaldehydeand (3) one-step sample preparation without heating. The present methodis particularly suitable for detecting residual formaldehyde in vaccinescontaining an adjuvant such as aluminum hydroxide. The method wasvalidated in terms of specificity, linearity, accuracy, repeatability,robustness and stability according to the International Conference onHarmonization (ICH) Q2 guidelines. In some embodiments 50 parts of thecomposition comprising a virus which has been treated with formaldehydeare mixed with 1 part of 15 to 25% (v/v) phosphoric acid and 2.5 partsof 0.9 to 1.1 mg/ml DNPH. In some embodiments 50 parts of thecomposition comprising a virus which has been treated with formaldehydeare mixed with 1 part of 20% (v/v) phosphoric acid and 2.5 parts of 1.0mg/ml DNPH.

In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated at a temperature of 18° C. to 30° C. In some embodimentsthe mixture of the composition comprising a virus which has been treatedwith formaldehyde with phosphoric acid and DNPH is incubated at atemperature of 20° C. to 25° C. In some embodiments the mixture of thecomposition comprising a virus which has been treated with formaldehydewith phosphoric acid and DNPH is incubated at a temperature of 22° C.

In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated for 10 to 30 minutes. In some embodiments the mixture ofthe composition comprising a virus which has been treated withformaldehyde with phosphoric acid and DNPH is incubated for 15 to 25minutes. In some embodiments the mixture of the composition comprising avirus which has been treated with formaldehyde with phosphoric acid andDNPH is incubated for 20 minutes.

In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated at a temperature of 18° C. to 30° C. for 10 to 30 minutes.In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated at a temperature of 18° C. to 30° C. for 15 to 25 minutes.In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated at a temperature of 18° C. to 30° C. for 20 minutes.

In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated at a temperature of 20° C. to 25° C. for 10 to 30 minutes.In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated at a temperature of 20° C. to 25° C. for 15 to 25 minutes.In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated at a temperature of 20° C. to 25° C. for 20 minutes.

In some embodiments the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHis incubated at a temperature of 22° C. for 10 to 30 minutes. In someembodiments the mixture of the composition comprising a virus which hasbeen treated with formaldehyde with phosphoric acid and DNPH isincubated at a temperature of 22° C. for 15 to 25 minutes. In someembodiments the mixture of the composition comprising a virus which hasbeen treated with formaldehyde with phosphoric acid and DNPH isincubated at a temperature of 22° C. for 20 minutes.

In some embodiments the mixture of 50 parts of the compositioncomprising a virus which has been treated with formaldehyde with 1 partof 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at atemperature of 18° C. to 30° C. for 10 to 30 minutes. In someembodiments the mixture of 50 parts of the composition comprising avirus which has been treated with formaldehyde with 1 part of 20%phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at atemperature of 18° C. to 30° C. for 15 to 25 minutes. In someembodiments the mixture of 50 parts of the composition comprising avirus which has been treated with formaldehyde with 1 part of 20%phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at atemperature of 18° C. to 30° C. for 20 minutes.

In some embodiments the mixture of 50 parts of the compositioncomprising a virus which has been treated with formaldehyde with 1 partof 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at atemperature of 20° C. to 25° C. for 10 to 30 minutes. In someembodiments the mixture of 50 parts of the composition comprising avirus which has been treated with formaldehyde with 1 part of 20%phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at atemperature of 20° C. to 25° C. for 15 to 25 minutes. In someembodiments the mixture of 50 parts of the composition comprising avirus which has been treated with formaldehyde with 1 part of 20%phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at atemperature of 20° C. to 25° C. for 20 minutes.

In some embodiments the mixture of 50 parts of the compositioncomprising a virus which has been treated with formaldehyde with 1 partof 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH is incubated at atemperature of 22° C. for 10 to 30 minutes. In some embodiments themixture of 50 parts of the composition comprising a virus which has beentreated with formaldehyde with 1 part of 20% phosphoric acid and 2.5parts of 1.0 mg/ml DNPH is incubated at a temperature of 22° C. for 15to 25 minutes. In some embodiments the mixture of 50 parts of thecomposition comprising a virus which has been treated with formaldehydewith 1 part of 20% phosphoric acid and 2.5 parts of 1.0 mg/ml DNPH isincubated at a temperature of 22° C. for 20 minutes.

After incubation, the mixture of the composition comprising a viruswhich has been treated with formaldehyde with phosphoric acid and DNPHmay be analyzed by any suitable method. In one embodiment, afterincubation, the mixture of the composition comprising a virus which hasbeen treated with formaldehyde with phosphoric acid and DNPH is analyzedby HPLC. In one embodiment, after incubation, the mixture of thecomposition comprising a virus which has been treated with formaldehydewith phosphoric acid and DNPH is analyzed by reverse phase HPLC. In oneembodiment, the ligand of the reversed phase HPLC column is selectedfrom C18, n-butal, n-octyl, phenyl and cyanopropyl. In one embodiment,the ligand of the reversed phase HPLC column is C18. In one embodiment,a mixture of water and acetonitrile (1:1, v/v) is used as the mobilephase in the reversed phase HPLC. In one embodiment, the detectionwavelength is 360 nm.

In one embodiment, the present disclosure provides a method fordetermining the residual formaldehyde content in a vaccine orimmunogenic composition comprising an inactivated virus, comprising thesteps of:

-   -   (a) providing a composition comprising a virus which has been        treated with formaldehyde;    -   (b) mixing 50 parts of the composition of (a) with 1 part of 20%        phosphoric acid and 2.5 parts of 1 mg/ml        2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;    -   (c) incubating the mixture of (b) for 20 minutes at room        temperature; and    -   (d) analyzing the mixture for the presence of residual        formaldehyde by reversed phase HPLC using a C18 column and a        mixture of water and acetonitrile (1:1, v/v) as the mobile        phase.

In one embodiment, the present disclosure provides a method fordetermining the residual formaldehyde content in a vaccine orimmunogenic composition comprising an inactivated Zika virus, comprisingthe steps of:

-   -   (a) providing a composition comprising a Zika virus which has        been treated with formaldehyde;    -   (b) mixing 50 parts of the composition of (a) with 1 part of 20%        phosphoric acid and 2.5 parts of 1 mg/ml        2,4-dinitrophenylhydrazine (DNPH), thereby providing a mixture;    -   (c) incubating the mixture of (b) for 20 minutes at room        temperature; and    -   (d) analyzing the mixture for the presence of residual        formaldehyde by reversed phase HPLC using a C18 column and a        mixture of water and acetonitrile (1:1, v/v) as the mobile        phase.

Adjuvants

Other aspects of the present disclosure relate to Zika virus vaccinesand/or immunogenic compositions containing one or more antigens from atleast one Zika virus described herein in combination with one or moreadjuvants. Such adjuvanted vaccines and/or immunogenic compositions ofthe present disclosure may be useful for treating or preventing Zikavirus infection in a human subject in need thereof and/or inducing animmune response, such as a protective immune response, against Zikavirus in a human subject in need thereof.

Various methods of achieving an adjuvant effect for vaccines are knownand may be used in conjunction with the Zika virus vaccines and/orimmunogenic compositions disclosed herein. General principles andmethods are detailed in “The Theory and Practical Application ofAdjuvants”, 1995, Duncan E. S. Stewart-Tull (ed.), John Wiley & SonsLtd, ISBN 0-471-95170-6, and also in “Vaccines: New GenerationImmunological Adjuvants”, 1995, Gregoriadis G et al. (eds.), PlenumPress, New York, ISBN 0-306-45283-9.

In some embodiments, a Zika virus vaccine or immunogenic compositionincludes the antigens and an adjuvant. Antigens may be in a mixture withat least one adjuvant, at a weight-based ratio of from about 10:1 toabout 10¹⁰:1 antigen:adjuvant, e.g., from about 10:1 to about 100:1,from about 100:1 to about 10³:1, from about 10³:1 to about 10⁴:1, fromabout 10⁴:1 to about 10⁵:1, from about 10⁵:1 to about 10⁶:1, from about10⁶:1 to about 10⁷:1, from about 10⁷:1 to about 10⁸:1, from about 10⁸:1to about 10⁹:1, or from about 10⁹:1 to about 10¹⁰:1 antigen:adjuvant.One of skill in the art can readily determine the appropriate ratiothrough information regarding the adjuvant and routine experimentationto determine optimal ratios.

Exemplary adjuvants may include, but are not limited to, aluminum salts,calcium phosphate, toll-like receptor (TLR) agonists, monophosphoryllipid A (MLA), MLA derivatives, synthetic lipid A, lipid A mimetics oranalogs, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpGoligos, lipopolysaccharide (LPS) of gram-negative bacteria,polyphosphazenes, emulsions (oil emulsions), chitosan, vitamin D,stearyl or octadecyl tyrosine, virosomes, cochleates,poly(lactide-co-glycolides) (PLG) microparticles, poloxamer particles,microparticles, liposomes, Complete Freund's Adjuvant (CFA), andIncomplete Freund's Adjuvant (IFA). In some embodiments, the adjuvant isan aluminum salt.

In some embodiments, the adjuvant includes at least one of alum,aluminum phosphate, aluminum hydroxide, potassium aluminum sulfate, andAlhydrogel 85. In some embodiments, aluminum salt adjuvants of thepresent disclosure have been found to increase adsorption of theantigens of the Zika virus vaccines and/or immunogenic compositions ofthe present disclosure. Accordingly, in some embodiments, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 91%, at least about 92%, at least about 93%, at least about94%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or about 100% of the antigen isadsorbed to the aluminum salt adjuvant.

In some embodiments, the vaccine or immunogenic composition includes analuminum salt adjuvant (e.g., alum) from about 100 μg to about 600 μg,from about 100 μg to about 500 μg, from about 125 μg to about 500 μg,from about 150 μg to about 500 μg, from about 175 μg to about 500 μg,from about 100 μg to about 450 μg, from about 125 μg to about 450 μg,from about 150 μg to about 450 μg, from about 175 μg to about 450 μg,from about 100 μg to about 400 μg, from about 125 μg to about 400 μg,from about 150 μg to about 400 μg, from about 175 μg to about 400 μg,from about 100 μg to about 350 μg, from about 125 μg to about 350 μg,from about 150 μg to about 350 μg, from about 175 μg to about 350 μg,from about 100 μg to about 300 μg, from about 125 μg to about 300 μg,from about 150 μg to about 300 μg, from about 175 μg to about 300 μg,from about 100 μg to about 250 μg, from about 125 μg to about 250 μg,from about 150 μg to about 250 μg, from about 175 μg to about 250 μg,from about 100 μg to about 225 μg, from about 125 μg to about 225 μg,from about 150 μg to about 225 μg, from about 175 μg to about 225 μg, orabout 200 μg. In some embodiments the vaccine or immunogenic compositionincludes an aluminum salt adjuvant (e.g., alum such as aluminumhydroxide) at about 100 μg to about 600 μg at about 100 μg to about 300μg or about 150 μg to about 250 μg or about 200 μg.

In some embodiments, the vaccines and/or immunogenic compositionscontains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purifiedinactivated whole Zika virus such as a Zika virus with a mutation whichis a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 orat a position corresponding to position 98 of SEQ ID NO:1 as describedherein in combination with one or more adjuvants, such as 100 μg toabout 600 μg or about 150 μg to about 250 μg or about 200 μg alum, suchas aluminum hydroxide.

In some embodiments, the vaccine or immunogenic composition contains adose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivatedwhole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ IDNO: 1, or at a position corresponding to position 98 of SEQ ID NO:1,wherein the Zika virus is derived from strain PRVABC59 in combinationwith one or more adjuvants, such as 100 μg to about 600 μg or about 150μg to about 250 μg or about 200 μg alum, such as aluminum hydroxide.

In some embodiments, the vaccine or immunogenic composition contains adose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivatedwhole Zika virus comprising a Trp98Gly mutation at position 98 of SEQ IDNO: 1, or at a position corresponding to position 98 of SEQ ID NO:1,wherein the Zika virus is derived from strain PRVABC59 comprising thegenomic sequence according to SEQ ID NO:2 in combination with one ormore adjuvants, such as 100 μg to about 600 μg or about 150 μg to about250 μg or about 200 μg alum, such as aluminum hydroxide.

In some embodiments, the vaccine or immunogenic composition contains adose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purified inactivatedwhole plaque purified Zika virus isolate comprising a Trp98Gly mutationat position 98 of SEQ ID NO: 1, or at a position corresponding toposition 98 of SEQ ID NO:1, wherein the Zika virus is derived fromstrain PRVABC59 comprising the genomic sequence according to SEQ ID NO:2in combination with one or more adjuvants, such as 100 μg to about 600μg or about 150 μg to about 250 μg or about 200 μg alum, such asaluminum hydroxide.

Certain embodiments of the present disclosure include a method forpreparing an adjuvanted Zika virus vaccine or immunogenic composition,which involves (a) mixing the vaccine or immunogenic composition with analuminum salt adjuvant, with the vaccine or immunogenic compositionincluding one or more antigens from at least one Zika virus describedherein and (b) incubating the mixture under suitable conditions for aperiod of time that ranges from about 1 hour to about 24 hours (e.g.,about 16 hours to about 24 hours), with at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99%, or about 100% of the antigen adsorbed to thealuminum salt adjuvant. In certain embodiments of the method, the atleast one Zika virus is a Zika virus comprising a non-human celladaptation mutation (e.g., a non-human cell adaptation mutation inprotein NS1 such as a Trp98Gly mutation.

In some embodiments of the method, the mixture is incubated at atemperature that ranges from about 2° C. to about 8° C. In someembodiments of the method, the mixture is incubated under constantmixing using any suitable mixer known in the art. In some embodiments ofthe method, the mixture is incubated at pH that ranges in value fromabout 6.5 to about 8.5, from about 6.5 to about 8, from about 6.8 toabout 7.8, from about 6.9 to about 7.6, from about 7 to about 7.5, fromabout 6.8 to about 8.5, from about 6.9 to about 8.5, or from about 7 toabout 8.5. In certain preferred embodiments, the mixture is incubated ata neutral pH. In some embodiments of the method, the aluminum saltadjuvant is selected from alum, aluminum phosphate, aluminum hydroxide,potassium aluminum sulfate, and Alhydrogel 85.

Monophosphoryl Lipid A (MLA), a non-toxic derivative of lipid A fromSalmonella, is a potent TLR-4 agonist that has been developed as avaccine adjuvant (Evans et al. (2003) Expert Rev. Vaccines 2(2):219-229). In pre-clinical murine studies intranasal MLA has been shownto enhance secretory, as well as systemic, humoral responses (Baldridgeet al. (2000) Vaccine 18(22): 2416-2425; Yang et al. (2002) Infect.Immun 70(7): 3557-3565). It has also been proven to be safe andeffective as a vaccine adjuvant in clinical studies of greater than120,000 patients (Baldrick et al. (2002) Regul. Toxicol. Pharmacol.35(3): 398-413; Baldrick et al. (2004) J. Appl. Toxicol. 24(4):261-268). MLA stimulates the induction of innate immunity through theTLR-4 receptor and is thus capable of eliciting nonspecific immuneresponses against a wide range of infectious pathogens, including bothgram negative and gram positive bacteria, viruses, and parasites(Baldrick et al. (2004) J. Appl. Toxicol. 24(4): 261-268; Persing et al.(2002) Trends Microbiol. 10(10 Suppl): S32-37). Inclusion of MLA inintranasal formulations should provide rapid induction of innateresponses, eliciting nonspecific immune responses from viral challengewhile enhancing the specific responses generated by the antigeniccomponents of the vaccine.

Accordingly, in one embodiment, the present disclosure provides acomposition comprising monophosphoryl lipid A (MLA), 3 De-O-acylatedmonophosphoryl lipid A (3D-MLA), or a derivative thereof as an enhancerof adaptive and innate immunity. Chemically 3D-MLA is a mixture of 3De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains. Apreferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed inEuropean Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA). Inanother embodiment, the present disclosure provides a compositioncomprising synthetic lipid A, lipid A mimetics or analogs, such asBioMira's PET Lipid A, or synthetic derivatives designed to functionlike TLR-4 agonists.

Additional exemplary adjuvants include, without limitation, polypeptideadjuvants that may be readily added to the antigens described herein byco-expression with the polypeptide components or fusion with thepolypeptide components to produce chimeric polypeptides. Bacterialflagellin, the major protein constituent of flagella, is an adjuvantwhich has received increasing attention as an adjuvant protein becauseof its recognition by the innate immune system by the toll-like receptorTLR5. Flagellin signaling through TLR5 has effects on both innate andadaptive immune functions by inducing DC maturation and migration aswell as activation of macrophages, neutrophils, and intestinalepithelial cells resulting in production of pro-inflammatory mediators.

TLR5 recognizes a conserved structure within flagellin monomers that isunique to this protein and is required for flagellar function,precluding its mutation in response to immunological pressure. Thereceptor is sensitive to a 100 fM concentration but does not recognizeintact filaments. Flagellar disassembly into monomers is required forbinding and stimulation.

As an adjuvant, flagellin has potent activity for induction ofprotective responses for heterologous antigens administered eitherparenterally or intranasally and adjuvant effects for DNA vaccines havealso been reported. A Th2 bias is observed when flagellin is employedwhich would be appropriate for a respiratory virus such as influenza butno evidence for IgE induction in mice or monkeys has been observed. Inaddition, no local or systemic inflammatory responses have been reportedfollowing intranasal or systemic administration in monkeys. The Th2character of responses elicited following use of flagellin is somewhatsurprising since flagellin signals through TLR5 in a MyD88-dependentmanner and all other MyD88-dependent signals through TLRs have beenshown to result in a Th1 bias. Importantly, pre-existing antibodies toflagellin have no appreciable effect on adjuvant efficacy making itattractive as a multi-use adjuvant.

A common theme in many recent intranasal vaccine trials is the use ofadjuvants and/or delivery systems to improve vaccine efficacy. In onesuch study an influenza H3 vaccine containing a genetically detoxifiedE. coli heat-labile enterotoxin adjuvant (LT R192G) resulted inheterosubtypic protection against H5 challenge but only followingintranasal delivery. Protection was based on the induction of crossneutralizing antibodies and demonstrated important implications for theintranasal route in development of new vaccines.

Cytokines, colony-stimulating factors (e.g., GM-CSF, CSF, and the like);tumor necrosis factor; interleukin-2, -7, -12, interferons and otherlike growth factors, may also be used as adjuvants as they may bereadily included in the Zika virus vaccines or immunogenic compositionsby admixing or fusion with the polypeptide component.

In some embodiments, the Zika virus vaccine and/or immunogeniccompositions disclosed herein may include other adjuvants that actthrough a Toll-like receptor such as a nucleic acid TLR9 ligandcomprising a 5′-TCG-3′ sequence; an imidazoquinoline TLR7 ligand; asubstituted guanine TLR7/8 ligand; other TLR7 ligands such asLoxoribine, 7-deazadeoxyguanosine, 7-thia-8-oxodeoxyguanosine, Imiquimod(R-837), and Resiquimod (R-848).

Certain adjuvants facilitate uptake of the vaccine molecules by APCs,such as dendritic cells, and activate these. Non-limiting examples areselected from the group consisting of an immune targeting adjuvant; animmune modulating adjuvant such as a toxin, a cytokine, and amycobacterial derivative; an oil formulation; a polymer; a micelleforming adjuvant; a saponin; an immunostimulating complex matrix (ISCOMmatrix); a particle; DDA; aluminum adjuvants; DNA adjuvants; MLA; and anencapsulating adjuvant.

Additional examples of adjuvants include agents such as aluminum saltssuch as hydroxide or phosphate (alum), commonly used as 0.05 to 0.1percent solution in buffered saline (see, e.g., Nicklas (1992) Res.Immunol. 143:489-493), admixture with synthetic polymers of sugars (e.g.Carbopol®) used as 0.25 percent solution, aggregation of the protein inthe vaccine by heat treatment with temperatures ranging between 70° C.to 101° C. for 30 second to 2 minute periods respectively and alsoaggregation by means of cross-linking agents are possible. Aggregationby reactivation with pepsin treated antibodies (Fab fragments) toalbumin, mixture with bacterial cells such as C. parvum or endotoxins orlipopolysaccharide components of gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide mono-oleate(Aracel A) or emulsion with 20 percent solution of a perfluorocarbon(Fluosol-DA) used as a block substitute may also be employed. Admixturewith oils such as squalene and IFA may also be used.

DDA (dimethyldioctadecylammonium bromide) is an interesting candidatefor an adjuvant, but also Freund's complete and incomplete adjuvants aswell as quillaja saponins such as QuilA and QS21 are interesting.Further possibilities include poly[di(earboxylatophenoxy)phosphazene(PCPP) derivatives of lipopolysaccharides such as monophosphoryl lipid A(MLA), muramyl dipeptide (MDP) and threonyl muramyl dipeptide (tMDP).The lipopolysaccharide based adjuvants may also be used for producing apredominantly Th1-type response including, for example, a combination ofmonophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A,together with an aluminum salt.

Liposome formulations are also known to confer adjuvant effects, andtherefore liposome adjuvants may be used in conjunction with the Zikavirus vaccines and/or immunogenic compositions.

Immunostimulating complex matrix type (ISCOM® matrix) adjuvants may alsobe used with the Zika virus vaccine antigens and immunogeniccompositions, especially since it has been shown that this type ofadjuvants are capable of up-regulating MHC Class II expression by APCs.An ISCOM matrix consists of (optionally fractionated) saponins(triterpenoids) from Quillaja saponaria, cholesterol, and phospholipid.When admixed with the immunogenic protein such as the Zika virus vaccineor immunogenic composition antigens, the resulting particulateformulation is what is known as an ISCOM particle where the saponin mayconstitute 60-70% w/w, the cholesterol and phospholipid 10-15% w/w, andthe protein 10-15% w/w. Details relating to composition and use ofimmunostimulating complexes can for example be found in theabove-mentioned text-books dealing with adjuvants, but also Morein B etal. (1995) Clin. Immunother. 3: 461-475 as well as Barr I G and MitchellG F (1996) Immunol. and Cell Biol. 74: 8-25 provide useful instructionsfor the preparation of complete immunostimulating complexes.

The saponins, whether or not in the form of iscoms, that may be used inthe adjuvant combinations with the Zika virus vaccines and immunogeniccompositions disclosed herein include those derived from the bark ofQuillaja Saponaria Molina, termed Quil A, and fractions thereof,described in U.S. Pat. No. 5,057,540 and “Saponins as vaccineadjuvants”, Kensil, C. R. (1996) Crit Rev Ther Drug Carrier Syst 12(1-2):1-55; and EP 0 362 279 B1. Exemplary fractions of Quil A are QS21,QS7, and QS17.

β-Escin is another hemolytic saponins for use in the adjuvantcompositions of the Zika virus vaccines and/or immunogenic compositions.Escin is described in the Merck index (12th ed: entry 3737) as a mixtureof saponins occurring in the seed of the horse chestnut tree, Lat:Aesculus hippocastanum. Its isolation is described by chromatography andpurification (Fiedler, Arzneimittel-Forsch. 4, 213 (1953)), and byion-exchange resins (Erbring et al., U.S. Pat. No. 3,238,190). Fractionsof escin have been purified and shown to be biologically active(Yoshikawa M, et al. (Chem Pharm Bull (Tokyo) 1996 August;44(8):1454-1464)). β-escin is also known as aescin.

Another hemolytic saponin for use in the Zika virus vaccines and/orimmunogenic compositions is Digitonin. Digitonin is described in theMerck index (12th Edition, entry 3204) as a saponin, being derived fromthe seeds of Digitalis purpurea and purified according to the proceduredescribed Gisvold et al. (1934) J. Am. Pharm. Assoc. 23: 664; andRuhenstroth-Bauer (1955) Physiol. Chem., 301, 621. Its use is describedas being a clinical reagent for cholesterol determination.

Another interesting possibility of achieving adjuvant effect is toemploy the technique described in Gosselin et al., 1992. In brief, thepresentation of a relevant antigen such as an antigen in a Zika virusvaccine and/or immunogenic composition of the present disclosure can beenhanced by conjugating the antigen to antibodies (or antigen bindingantibody fragments) against the FC receptors on monocytes/macrophages.Especially conjugates between antigen and anti-FCRI have beendemonstrated to enhance immunogenicity for the purposes of vaccination.The antibody may be conjugated to the Zika virus vaccine or immunogeniccomposition antigens after generation or as a part of the generationincluding by expressing as a fusion to any one of the polypeptidecomponents of the Zika virus vaccine and/or immunogenic compositionantigens. Other possibilities involve the use of the targeting andimmune modulating substances (e.g., cytokines). In addition, syntheticinducers of cytokines such as poly I:C may also be used.

Suitable mycobacterial derivatives may be selected from the groupconsisting of muramyl dipeptide, complete Freund's adjuvant, RIBI, (RibiImmunoChem Research Inc., Hamilton, Mont.) and a diester of trehalosesuch as TDM and TDE.

Examples of suitable immune targeting adjuvants include CD40 ligand andCD40 antibodies or specifically binding fragments thereof (cf. thediscussion above), mannose, a Fab fragment, and CTLA-4.

Examples of suitable polymer adjuvants include a carbohydrate such asdextran, PEG, starch, mannan, and mannose; a plastic polymer; and latexsuch as latex beads.

Yet another interesting way of modulating an immune response is toinclude the immunogen (optionally together with adjuvants andpharmaceutically acceptable carriers and vehicles) in a “virtual lymphnode” (VLN) (a proprietary medical device developed by ImmunoTherapy,Inc., 360 Lexington Avenue, New York, N.Y. 10017-6501). The VLN (a thintubular device) mimics the structure and function of a lymph node.Insertion of a VLN under the skin creates a site of sterile inflammationwith an upsurge of cytokines and chemokines. T- and B-cells as well asAPCs rapidly respond to the danger signals, home to the inflamed siteand accumulate inside the porous matrix of the VLN. It has been shownthat the necessary antigen dose required to mount an immune response toan antigen is reduced when using the VLN, and that immune protectionconferred by vaccination using a VLN surpassed conventional immunizationusing Ribi as an adjuvant. The technology is described briefly in GelberC et al., 1998, “Elicitation of Robust Cellular and Humoral ImmuneResponses to Small Amounts of Immunogens Using a Novel Medical DeviceDesignated the Virtual Lymph Node”, in: “From the Laboratory to theClinic, Book of Abstracts, Oct. 12-15, 1998, Seascape Resort, Aptos,Calif.”

Oligonucleotides may be used as adjuvants in conjunction with the Zikavirus vaccine and/or immunogenic composition antigens and may containtwo or more dinucleotide CpG motifs separated by at least three or moreor even at least six or more nucleotides. CpG-containingoligonucleotides (in which the CpG dinucleotide is unmethylated) inducea predominantly Th1 response. Such oligonucleotides are well known andare described, for example, in WO 96/02555, WO 99/33488 and U.S. Pat.Nos. 6,008,200 and 5,856,462.

Such oligonucleotide adjuvants may be deoxynucleotides. In certainembodiments, the nucleotide backbone in the oligonucleotide isphosphorodithioate, or a phosphorothioate bond, although phosphodiesterand other nucleotide backbones such as PNA including oligonucleotideswith mixed backbone linkages may also be used. Methods for producingphosphorothioate oligonucleotides or phosphorodithioate are described inU.S. Pat. Nos. 5,666,153, 5,278,302 and WO 95/26204.

Exemplary oligonucleotides have the following sequences. The sequencesmay contain phosphorothioate modified nucleotide backbones:

OLIGO 1:  (SEQ ID NO: 3) TCC ATG ACG TTC CTG ACG TT (CpG 1826);OLIGO 2:  (SEQ ID NO: 4) TCT CCC AGC GTG CGC CAT (CpG 1758);  OLIGO 3: (SEQ ID NO: 5) ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG; OLIGO 4: (SEQ ID NO: 6) TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006); and OLIGO 5:  (SEQ ID NO: 7) TCC ATG ACG TTC CTG ATG CT (CpG 1668) 

Alternative CpG oligonucleotides include the above sequences withinconsequential deletions or additions thereto. The CpG oligonucleotidesas adjuvants may be synthesized by any method known in the art (e.g., EP468520). For example, such oligonucleotides may be synthesized utilizingan automated synthesizer. Such oligonucleotide adjuvants may be between10-50 bases in length. Another adjuvant system involves the combinationof a CpG-containing oligonucleotide and a saponin derivativeparticularly the combination of CpG and QS21 is disclosed in WO00/09159.

Many single or multiphase emulsion systems have been described. One ofskill in the art may readily adapt such emulsion systems for use with aZika virus vaccine and/or immunogenic composition antigens so that theemulsion does not disrupt the antigen's structure. Oil in water emulsionadjuvants per se have been suggested to be useful as adjuvantcompositions (EP 399 843B), also combinations of oil in water emulsionsand other active agents have been described as adjuvants for vaccines(WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241). Other oil emulsionadjuvants have been described, such as water in oil emulsions (U.S. Pat.No. 5,422,109; EP 0 480 982 B2) and water in oil in water emulsions(U.S. Pat. No. 5,424,067; EP 0 480 981 B).

The oil emulsion adjuvants for use with the Zika virus vaccines and/orimmunogenic compositions described herein may be natural or synthetic,and may be mineral or organic. Examples of mineral and organic oils willbe readily apparent to one skilled in the art.

In order for any oil in water composition to be suitable for humanadministration, the oil phase of the emulsion system may include ametabolizable oil. The meaning of the term metabolizable oil is wellknown in the art. Metabolizable can be defined as “being capable ofbeing transformed by metabolism” (Dorland's Illustrated MedicalDictionary, W.B. Sanders Company, 25th edition (1974)). The oil may beany vegetable oil, fish oil, animal oil or synthetic oil, which is nottoxic to the recipient and is capable of being transformed bymetabolism. Nuts (such as peanut oil), seeds, and grains are commonsources of vegetable oils. Synthetic oils may also be used and caninclude commercially available oils such as NEOBEE® and others. Squalene(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is anunsaturated oil which is found in large quantities in shark-liver oil,and in lower quantities in olive oil, wheat germ oil, rice bran oil, andyeast, and may be used with the Zika virus vaccine and/or immunogeniccompositions. Squalene is a metabolizable oil virtue of the fact that itis an intermediate in the biosynthesis of cholesterol (Merck index, 10thEdition, entry no. 8619).

Exemplary oil emulsions are oil in water emulsions, and in particularsqualene in water emulsions.

In addition, the oil emulsion adjuvants for use with the Zika virusvaccine and/or immunogenic compositions may include an antioxidant, suchas the oil α-tocopherol (vitamin E, EP 0 382 271 B1).

WO 95/17210 and WO 99/11241 disclose emulsion adjuvants based onsqualene, α-tocopherol, and TWEEN 80™, optionally formulated with theimmunostimulants QS21 and/or 3D-MLA. WO 99/12565 discloses animprovement to these squalene emulsions with the addition of a sterolinto the oil phase. Additionally, a triglyceride, such as tricaprylin(C27H5006), may be added to the oil phase in order to stabilize theemulsion (WO 98/56414).

The size of the oil droplets found within the stable oil in wateremulsion may be less than 1 micron, may be in the range of substantially30-600 nm, substantially around 30-500 nm in diameter, or substantially150-500 nm in diameter, and in particular about 150 nm in diameter asmeasured by photon correlation spectroscopy. In this regard, 80% of theoil droplets by number may be within these ranges, more than 90% or morethan 95% of the oil droplets by number are within the defined sizeranges. The amounts of the components present in oil emulsions areconventionally in the range of from 2 to 10% oil, such as squalene; andwhen present, from 2 to 10% alpha tocopherol; and from 0.3 to 3%surfactant, such as polyoxyethylene sorbitan monooleate. The ratio ofoil:alpha tocopherol may be equal or less than 1 as this provides a morestable emulsion. SPAN 85™ may also be present at a level of about 1%. Insome cases it may be advantageous that the Zika virus vaccines and/orimmunogenic compositions disclosed herein will further contain astabilizer.

The method of producing oil in water emulsions is well known to oneskilled in the art. Commonly, the method includes the step of mixing theoil phase with a surfactant such as a PBS/TWEEN80® solution, followed byhomogenization using a homogenizer, it would be clear to one skilled inthe art that a method comprising passing the mixture twice through asyringe needle would be suitable for homogenizing small volumes ofliquid. Equally, the emulsification process in microfluidizer (M110Smicrofluidics machine, maximum of 50 passes, for a period of 2 minutesat maximum pressure input of 6 bar (output pressure of about 850 bar))could be adapted by one skilled in the art to produce smaller or largervolumes of emulsion. This adaptation could be achieved by routineexperimentation comprising the measurement of the resultant emulsionuntil a preparation was achieved with oil droplets of the requireddiameter.

Alternatively the Zika virus vaccines and/or immunogenic compositionsmay be combined with vaccine vehicles composed of chitosan (as describedabove) or other polycationic polymers, polylactide andpolylactide-coglycolide particles, poly-N-acetyl glucosamine-basedpolymer matrix, particles composed of polysaccharides or chemicallymodified polysaccharides, liposomes and lipid-based particles, particlescomposed of glycerol monoesters, etc. The saponins may also beformulated in the presence of cholesterol to form particulate structuressuch as liposomes or ISCOMs. Furthermore, the saponins may be formulatedtogether with a polyoxyethylene ether or ester, in either anon-particulate solution or suspension, or in a particulate structuresuch as a paucilamelar liposome or ISCOM.

Additional illustrative adjuvants for use in the Zika virus vaccinesand/or immunogenic compositions as described herein include SAF (Chiron,Calif., United States), MF-59 (Chiron, see, e.g., Granoff et al. (1997)Infect Immun 65 (5):1710-1715), the SBAS series of adjuvants (e.g.,SB-AS2 (an oil-in-water emulsion containing MLA and QS21); SBAS-4(adjuvant system containing alum and MLA), available from SmithKlineBeecham, Rixensart, Belgium), Detox (Enhanzyn®) (GlaxoSmithKline),RC-512, RC-522, RC-527, RC-529, RC-544, and RC-560 (GlaxoSmithKline) andother aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720.

Other examples of adjuvants include, but are not limited to, Hunter'sTiterMax® adjuvants (CytRx Corp., Norcross, Ga.); Gerbu adjuvants (GerbuBiotechnik GmbH, Gaiberg, Germany); nitrocellulose (Nilsson and Larsson(1992) Res. Immunol. 143:553-557); alum (e.g., aluminum hydroxide,aluminum phosphate) emulsion based formulations including mineral oil,non-mineral oil, water-in-oil or oil-in-water emulsions, such as theSeppic ISA series of Montamide adjuvants (e.g., ISA-51, ISA-57, ISA-720,ISA-151, etc.; Seppic, Paris, France); and PROVAX® (IDECPharmaceuticals); OM-174 (a glucosamine disaccharide related to lipidA); Leishmania elongation factor; non-ionic block copolymers that formmicelles such as CRL 1005; and Syntex Adjuvant Formulation. See, e.g.,O'Hagan et al. (2001) Biomol Eng. 18(3):69-85; and “Vaccine Adjuvants:Preparation Methods and Research Protocols” D. O'Hagan, ed. (2000)Humana Press.

Other exemplary adjuvants include adjuvant molecules of the generalformula: HO(CH₂CH₂O)n-A-R, (I) where, n is 1-50, A is a bond or —C(O)—,R is C1-50 alkyl or Phenyl C1-50 alkyl.

One embodiment consists of a vaccine formulation comprising apolyoxyethylene ether of general formula (I), where n is between 1 and50, 4-24, or 9; the R component is C1-50, C4-C20 alkyl, or C12 alkyl,and A is a bond. The concentration of the polyoxyethylene ethers shouldbe in the range 0.1-20%, from 0.1-10%, or in the range 0.1-1%. Exemplarypolyoxyethylene ethers are selected from the following group:polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether,polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.Polyoxyethylene ethers such as polyoxyethylene lauryl ether aredescribed in the Merck index (12th edition: entry 7717). These adjuvantmolecules are described in WO 99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, anadjuvant combination may include the CpG as described above.

Further examples of suitable pharmaceutically acceptable excipients foruse with the Zika virus vaccines and/or immunogenic compositionsdisclosed herein include water, phosphate buffered saline, isotonicbuffer solutions.

Virus Purification

Further aspects of the present disclosure relate to methods of purifyingZika virus. In some embodiments, the method includes inoculating aplurality of cells with an inoculum containing a population of Zikaviruses, and obtaining from one or more of the inoculated cells a Zikavirus clonal isolate by plaque purification. In some embodiments, thecells are non-human cells (e.g., insect cells, mammalian cells, etc.).In some embodiments, the cells are insect cells (such as any of themosquito cells/cell lines described herein). In some embodiments, thecells are mammalian cells (such as any of the mammalian cells/cell linesdescribed herein). In some embodiments, the mammalian cells are monkeycells. In some embodiments, the mammalian cells are Vero cells.

In some embodiments, the population of Zika virus is heterogeneous(e.g., comprising two or more genotypes). The two or more genotypesdiffer from each other in at least one nucleotide. In some embodiments,the population of Zika viruses comprises a Zika virus clinical isolate(e.g., from strain PRVABC59) and/or one or more Zika viruses that havebeen previously passaged in cell culture. A clinical isolate of the Zikavirus is obtained from a sample of a patient who is infected with Zikavirus. In some embodiments, plaque purification (e.g., as describedherein) allows for the substantial and/or complete separation of a(genetically homogenous) clonal isolate from a heterogeneous viralpopulation. In some embodiments, the monkey cells are from a VERO cellline (e.g., VERO 10-87 cells). In some embodiments, the inoculumcomprises human serum. In some embodiments, the inoculum comprises oneor more adventitious agents (e.g., one or more contamination viruses).In some embodiments, plaque purification (e.g., as described herein)allows for the substantial and/or complete purification of a(genetically homogenous) clonal isolate away from one or moreadventitious agents.

In some embodiments, the methods described for isolating and/orpurifying a Zika virus clonal includes one or more (e.g., one or more,two or more, three or more, four or more, five or more, etc.) additionalplaque purifications of the Zika virus clonal isolate. In someembodiments, the methods described for isolating and/or purifying a Zikavirus clonal isolate includes passaging the Zika virus clonal isolateone or more (e.g., one or more, two or more, three or more, four ormore, five or more, etc.) times in cell culture (e.g., in insect cellssuch as a mosquito cell line and/or in mammalian cells such as a VEROcell line).

Further aspects of the present disclosure relate to methods of purifyingZika virus for the preparation of a vaccine or immunogenic composition.In some embodiments, the methods include one or more (e.g., one or more,two or more, three or more, four or more, five or more, or six) steps of(in any order, including the following order): performing depthfiltration of a sample or preparation containing a Zika virus; bufferexchanging and/or diluting a sample containing a Zika virus (e.g., bycross flow filtration (CFF)) to produce a retentate; binding a samplecomprising a Zika virus to an ion exchange membrane (e.g., an anionexchange membrane, a cation exchange membrane) to produce a boundfraction, where the bound fraction comprises the Zika virus, and elutingthe bound fraction from the ion exchange membrane; treating a samplecontaining a Zika virus with an effective amount of any of the chemicalinactivators described herein; neutralizing a sample containing achemically inactivated Zika virus with sodium metabisulfite; and/orpurifying a neutralized sample comprising a chemically inactivated Zikavirus (e.g., by cross flow filtration (CFF)). In some embodiments, themethod includes the steps of (a) passing a sample containing a Zikavirus through a first depth filter to produce a first eluate, where thefirst eluate contains the Zika virus; (b) buffer exchanging and/ordiluting the first eluate by cross flow filtration (CFF) to produce afirst retentate, where the first retentate contains the Zika virus; (c)binding the first retentate to an ion exchange membrane to produce afirst bound fraction, where the first bound fraction contains the Zikavirus, and eluting the first bound fraction from the ion exchangemembrane to produce a second eluate, where the second eluate containsthe Zika virus; (d) passing the second eluate through a second depthfilter to produce a second retentate, wherein the second retentatecontains the Zika virus; (e) treating the second retentate with aneffective amount of a chemical inactivator; (f) neutralizing the treatedsecond retentate with sodium metabisulfite; and (g) purifying theneutralized second retentate by cross flow filtration (CFF).

Depth filters may be applied in a cartridge or capsule format, such aswith the SUPRACAP™ series of depth filter capsules (Pall Corporation)using a Bio 20 SEITZ® depth filter sheet. Other suitable depthfiltration techniques and apparatuses are known in the art and includeSartorius PP3 filters. In some embodiments, the depth filter has a poresize of between about 0.2 μm and about 3 μm. In some embodiments, thepore size of the depth filter is less than about any of the followingpore sizes (in μm): 3, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8, 1.6, 1.4, 1.2, 1.0,0.8, 0.6, and 0.4. In some embodiments, the pore size of the depthfilter is greater than about any of the following pore sizes (in μm):0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, or 2.8.That is, the pore size of the depth filter can be any of a range of poresizes (in μm) having an upper limit of 3, 2.8, 2.6, 2.4, 2.2, 2.0, 1.8,1.6, 1.4, 1.2, 1.0, 0.8, 0.6, and 0.4 and an independently selectedlower limit of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2,2.4, 2.6, or 2.8; wherein the lower limit is less than the upper limit.

As described herein, cation exchange and anion exchange chromatographymay be used in the methods of the present disclosure to purify a Zikavirus harvested from a cell of the present disclosure. For example,clarified viral harvest may be basified, loaded onto an anion exchangemembrane, eluted by salt or pH, filtered, and inactivated. This is onlyan exemplary scheme, and one of skill in the art may readily contemplatevariants thereof with substituted, deleted, inserted, or reorderedsteps.

Anion and cation exchange chromatography both rely on the attraction ofcharged macromolecules of interest (e.g., a virus) in a mobile phase toa substrate having an opposite charge. In cation exchangechromatography, the negatively charged substrate or membrane attractspositively charged macromolecules. In anion exchange chromatography, thepositively charged substrate or membrane attracts negatively chargedmacromolecules. Once macromolecules are bound or loaded onto thesubstrate, they may be eluted in linear or step-wise fashion from thesubstrate in a manner dependent on their characteristics, therebyenacting a separation of differently charged molecules. This principlemay be used to purify viruses from other macromolecules. Elution may beeffected by varying pH or salt content of the mobile phase buffer.Elution may be gradient or step-wise. As described herein, elution maybe effected using a change in pH of the mobile phase or by using achange in ionic strength of the mobile phase (e.g., through addition ofa salt). A variety of salts are used for elution, including withoutlimitation sodium chloride, potassium chloride, sodium sulphate,potassium sulphate, ammonium sulphate, sodium acetate, potassiumphosphate, calcium chloride, and magnesium chloride. In certainembodiments, the salt is NaCl. A variety of suitable buffers are knownin the art and described herein. Viral purification methods using ionexchange chromatography are also generally known; see, e.g.,purification of influenza virus available online atwww.pall.com/pdfs/Biopharmaceuticals/MustangQXT_AcroPrep_USD2916.pdf.

A variety of devices known in the art are suitable for cation exchangechromatography (optionally including filtration), such as the Mustang® Ssystem (Pall Corporation), which uses a cation exchange membrane with a0.65 μm pore size. A variety of functional groups are used for cationexchange membranes, including without limitation pendant sulfonicfunctional groups in a cross-linked, polymeric coating. A variety ofbuffers may be used to bind an eluate containing a Zika virus of thepresent disclosure to a cation exchange membrane. Exemplary buffersinclude, without limitation, citrate and phosphate buffers (additionalbuffers are described infra). In some embodiments, a buffer used incation exchange chromatography (e.g., in loading and/or elution)contains polysorbate (e.g., TWEEN®-80 at 0.05%, 0.1%, 0.25%, or 0.5%).

A variety of devices known in the art are suitable for anion exchangechromatography (optionally including filtration), such as the Mustang® Qsystem (Pall Corporation), which uses an anion exchange membrane with a0.8 μm pore size. Another suitable anion exchange membrane is SartobindQIEXNano. A variety of functional groups are used for anion exchangemembranes, including without limitation pendant quaternary aminefunctional groups in a cross-linked, polymeric coating. A variety ofbuffers may be used to bind an eluate containing a Zika virus of thepresent disclosure to an anion exchange membrane. Exemplary buffersinclude, without limitation, phosphate buffer (additional buffers aredescribed infra). In some embodiments, a buffer used in anion exchangechromatography (e.g., in loading and/or elution) contains polysorbate(e.g., TWEEN®-80 at 0.05%, 0.1%, 0.25%, or 0.5%). In some embodiments,the virus is eluted by step elution, e.g. using 250 mM NaCl, 500 mM NaCland 750 mM NaCl.

Formulations and Dose of Vaccines and/or Immunogenic Compositions

Further aspects of the present disclosure relate to formulations ofvaccines and/or immunogenic compositions of the present disclosurecontaining one or more antigens from a Zika virus described herein.

Such vaccines and/or immunogenic compositions of the present disclosurecontaining one or more antigens from a Zika virus described herein maybe useful for treating or preventing Zika virus infection in a humansubject in need thereof and/or inducing an immune response, such as aprotective immune response, against Zika virus in a human subject inneed thereof.

Typically, vaccines and/or immunogenic compositions of the presentdisclosure are prepared as injectables either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. Such preparations mayalso be emulsified or produced as a dry powder. The active immunogenicingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, sucrose, glycerol,ethanol, or the like, and combinations thereof. In addition, if desired,the vaccine or immunogenic composition may contain auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents, or adjuvantswhich enhance the effectiveness of the vaccine or immunogeniccomposition.

Vaccines or immunogenic compositions may be conventionally administeredparenterally, by injection, for example, either subcutaneously,transcutaneously, intradermally, subdermally or intramuscularly. Incertain embodiments the composition is administered intramuscular orsubcutaneously. Additional formulations which are suitable for othermodes of administration include suppositories and, in some cases, oral,peroral, intranasal, buccal, sublingual, intraperitoneal, intravaginal,anal and intracranial formulations. For suppositories, traditionalbinders and carriers may include, for example, polyalkalene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, or even 1-2%. Incertain embodiments, a low melting wax, such as a mixture of fatty acidglycerides or cocoa butter is first melted and the Zika virus vaccineand/or immunogenic composition described herein is dispersedhomogeneously, for example, by stirring. The molten homogeneous mixtureis then poured into conveniently sized molds and allowed to cool and tosolidify.

Formulations suitable for intranasal delivery include liquids (e.g.,aqueous solution for administration as an aerosol or nasal drops) anddry powders (e.g. for rapid deposition within the nasal passage).Formulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, sucrose, trehalose, xylitol,and chitosan. Mucosadhesive agents such as chitosan can be used ineither liquid or powder formulations to delay mucociliary clearance ofintranasally-administered formulations. Sugars such as mannitol,sorbitol, trehalose, and/or sucrose can be used as stability agents inliquid formulations and as stability, bulking, or powder flow and sizeagents in dry powder formulations. In addition, adjuvants such asmonophosphoryl lipid A (MLA), or derivatives thereof, or CpGoligonucleotides can be used in both liquid and dry powder formulationsas an immunostimulatory adjuvant.

Formulations suitable for oral delivery include liquids, solids,semi-solids, gels, tablets, capsules, lozenges, and the like.Formulations suitable for oral delivery include tablets, lozenges,capsules, gels, liquids, food products, beverages, nutraceuticals, andthe like. Formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, sorbitol, trehalose, polyolssuch as sugars such as sucrose, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. OtherZika virus vaccines and immunogenic compositions may take the form ofsolutions, suspensions, pills, sustained release formulations or powdersand contain 10-95% of active ingredient, or 25-70%. For oralformulations, cholera toxin is an interesting formulation partner (andalso a possible conjugation partner).

The Zika virus vaccines and/or immunogenic compositions when formulatedfor vaginal administration may be in the form of pessaries, tampons,creams, gels, pastes, foams or sprays. Any of the foregoing formulationsmay contain agents in addition to Zika virus vaccine and/or immunogeniccompositions, such as carriers, known in the art to be appropriate.

In some embodiments, the Zika virus vaccines and/or immunogeniccompositions of the present disclosure may be formulated for systemic orlocalized delivery. Such formulations are well known in the art.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Systemic and localized routes of administration include, e.g.,intradermal, topical application, intravenous, intramuscular, etc.

The vaccines and/or immunogenic compositions of the present disclosuremay be administered in a manner compatible with the dosage formulation,and in such amount as will be therapeutically effective and immunogenic.The dosage of the antigen may range in particular from, about 1 μg toabout 100 μg, about 1 μg to about 40 μg, about 1 μg to about 30 μg,about 1 μg to about 20 μg, about 1 μg to about 15 μg, or from about 2 μgto about 15 μg, or from about 5 μg to about 15 μg, or from about 10 μgto about 15 μg, The dosage may in particular be about 2 μg, about 5 μg,about 10 μg, about 15 μg or about 20 μg, in particular about 10 μg. Theamount of the antigen, i.e. the purified inactivated Zika virus, can bedetermined by a Bradford assay (Bradford et al. (1976) Anal. Biochem.72: 248-254) using defined amounts of recombinant Zika envelope proteinto establish the standard curve. Thus, the dosage of the antigen mayalso be referred to as micrograms (μg) of Zika Envelope protein E (μgEnv). μg Antigen and μg Env thus mean the same within the meaning ofthis disclosure. In some embodiments, the vaccines and/or immunogeniccompositions contains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg ofantigen in the form of a purified inactivated whole Zika virus such as aZika virus with a mutation which is a tryptophan to glycine substitutionat position 98 of SEQ ID NO:1 or at a position corresponding to position98 of SEQ ID NO:1 as described herein.

In some embodiments, the vaccine or immunogenic composition contains adose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of antigen in the form ofa purified inactivated whole Zika virus comprising a Trp98Gly mutationat position 98 of SEQ ID NO: 1, or at a position corresponding toposition 98 of SEQ ID NO:1, wherein the Zika virus is derived fromstrain PRVABC59.

In some embodiments, the vaccine or immunogenic composition contains adose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of antigen in the form ofa purified inactivated whole Zika virus comprising a Trp98Gly mutationat position 98 of SEQ ID NO: 1, or at a position corresponding toposition 98 of SEQ ID NO:1, wherein the Zika virus is derived fromstrain PRVABC59 comprising the genomic sequence according to SEQ IDNO:2.

In some embodiments, the vaccine or immunogenic composition contains adose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of antigen in the form ofa purified inactivated whole plaque purified Zika virus isolatecomprising a Trp98Gly mutation at position 98 of SEQ ID NO: 1, or at aposition corresponding to position 98 of SEQ ID NO:1, wherein the Zikavirus is derived from strain PRVABC59 comprising the genomic sequenceaccording to SEQ ID NO:2.

In certain such embodiments the vaccine or immunogenic compositioncomprises

a dose of about 10 μg of purified inactivated whole virus

about 200 μg aluminum hydroxide,

a buffer; and optionally

a sugar such as sucrose.

Suitable regimens for initial administration and booster shots are alsovariable but are typified by an initial administration followed bysubsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine or immunogenic composition areapplicable. These include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like for e.g. intramuscular orsubcutaneous administration. The dosage of the vaccine or immunogeniccomposition will depend on the route of administration and may varyaccording to the age of the person to be vaccinated and the formulationof the antigen. The vaccine or immunogenic composition can have a unitdosage volume of more than 0.5 mL, of 0.5 mL or of less than 0.5 mL, asdescribed herein. For instance, it can be administered at a volume of0.25 mL. A volume of 0.5 mL are suitable for intramuscular orsubcutaneous administration.

Delivery agents that improve mucoadhesion can also be used to improvedelivery and immunogenicity especially for intranasal, oral or lungbased delivery formulations. One such compound, chitosan, theN-deacetylated form of chitin, is used in many pharmaceuticalformulations. It is an attractive mucoadhesive agent for intranasalvaccine delivery due to its ability to delay mucociliary clearance andallow more time for mucosal antigen uptake and processing. In addition,it can transiently open tight junctions which may enhancetransepithelial transport of antigen to the NALT. In a recent humantrial, a trivalent inactivated influenza vaccine administeredintranasally with chitosan but without any additional adjuvant yieldedseroconversion and HI titers that were only marginally lower than thoseobtained following intramuscular inoculation.

Chitosan can also be formulated with adjuvants that function wellintranasally such as the genetically detoxified E. coli heat-labileenterotoxin mutant LTK63. This adds an immunostimulatory effect on topof the delivery and adhesion benefits imparted by chitosan resulting inenhanced mucosal and systemic responses.

Finally, it should be noted that chitosan formulations can also beprepared in a dry powder format that has been shown to improve vaccinestability and result in a further delay in mucociliary clearance overliquid formulations. This was seen in a recent human clinical trialinvolving an intranasal dry powder diphtheria toxoid vaccine formulatedwith chitosan in which the intranasal route was as effective as thetraditional intramuscular route with the added benefit of secretory IgAresponses. The vaccine was also very well tolerated. Intranasal drypowdered vaccines for anthrax containing chitosan and MLA, orderivatives thereof, induce stronger responses in rabbits thanintramuscular inoculation and are also protective against aerosol sporechallenge.

Intranasal vaccines represent an exemplary formulation as they canaffect the upper and lower respiratory tracts in contrast toparenterally administered vaccines which are better at affecting thelower respiratory tract. This can be beneficial for inducing toleranceto allergen-based vaccines and inducing immunity for pathogen-basedvaccines.

In addition to providing protection in both the upper and lowerrespiratory tracts, intranasal vaccines avoid the complications ofneedle inoculations and provide a means of inducing both mucosal andsystemic humoral and cellular responses via interaction of particulateand/or soluble antigens with nasopharyngeal-associated lymphoid tissues(NALT).

Vaccines and/or immunogenic compositions of the present disclosure arepharmaceutically acceptable. They may include components in addition tothe antigen and adjuvant, e.g. they will typically include one or morepharmaceutical carrier(s) and/or excipient(s). A thorough discussion ofsuch components is available in Gennaro (2000) Remington: The Scienceand Practice of Pharmacy. 20th edition, ISBN: 0683306472.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Vaccines and/or immunogenic compositions of the present disclosure mayinclude one or more buffers. Typical buffers include: a phosphatebuffer; a Tris buffer; a borate buffer; a succinate buffer; a histidinebuffer (particularly with an aluminum hydroxide adjuvant); or a citratebuffer. Buffers will typically be included in the 5-20 mM range.

The pH of a vaccine or immunogenic composition will generally be between5.0 and 8.5 or 5.0 and 8.1, and more typically between 6.0 and 8.5 e.g.between 6.0 and 8.0, between 6.5 and 8.0, between 6.5 and 7.5, between7.0 and 8.5, between 7.0 and 8.0, or between 7.0 and 7.8. Amanufacturing process of the present disclosure may therefore include astep of adjusting the pH of the bulk vaccine prior to packaging.

The vaccine or immunogenic composition is preferably sterile. It ispreferably non pyrogenic, e.g. containing <1 EU (endotoxin unit, astandard measure) per dose, and preferably <0.1 EU per dose. It ispreferably gluten free.

In certain embodiments, the vaccines and/or immunogenic compositions ofthe present disclosure may include a detergent in an effectiveconcentration. In some embodiments, an effective amount of detergent mayinclude without limitation, about 0.00005% v/v to about 5% v/v or about0.0001% v/v to about 1% v/v. In certain embodiments, an effective amountof detergent is about 0.001% v/v, about 0.002% v/v, about 0.003% v/v,about 0.004% v/v, about 0.005% v/v, about 0.006% v/v, about 0.007% v/v,about 0.008% v/v, about 0.009% v/v, or about 0.01% v/v. Without wishingto be bound by theory, detergents help maintain the vaccines and/orimmunogenic compositions of the present disclosure in solution and helpto prevent the vaccines and/or immunogenic compositions fromaggregating.

Suitable detergents include, for example, polyoxyethylene sorbitan estersurfactant (known as ‘Tweens’), octoxynol (such as octoxynol-9 (Triton X100) or t-octylphenoxypolyethoxyethanol), cetyl trimethyl ammoniumbromide (‘CTAB’), and sodium deoxycholate, particularly for a split orsurface antigen vaccine. The detergent may be present only at traceamounts. Other residual components in trace amounts could be antibiotics(e.g. neomycin, kanamycin, polymyxin B). In some embodiments, thedetergent contains polysorbate. In some embodiments, the effectiveconcentration of detergent includes ranges from about 0.00005% v/v toabout 5% v/v.

The vaccines and/or immunogenic compositions are preferably stored atbetween 2° C. and 8° C. They should ideally be kept out of direct light.The antigen and emulsion will typically be in admixture, although theymay initially be presented in the form of a kit of separate componentsfor extemporaneous admixing. Vaccines and/or immunogenic compositionswill generally be in aqueous form when administered to a human subject.

Methods of the Present Disclosure

The present invention is in particular also directed to a method oftreating or preventing, in particular preventing Zika virus infectionand/or preventing Zika virus disease in a human subject in need thereofcomprising administering to a human subject or a human subjectpopulation a therapeutically effective amount of a vaccine orimmunogenic composition as described herein.

The disease is in general mild and of short duration. Some clinicalmanifestations include, but are not limited to, mild fever,maculopapular rash, conjunctivitis and arthralgia. Despite mild clinicalsymptoms in the pregnant woman, Zika virus infection during pregnancyhas been associated with serious outcomes for the fetus and newborn. Theseverity of the disease is related to the consequences in the fetus andnewborn child from women with Zika virus infection during pregnancy. Thespectrum of congenital anomalies associated with Zika virus infection,known as Congenital Zika Syndrome (CZS), consists of severe microcephalywith partially collapsed skull, cerebral cortices with subcorticalcalcifications, macular scarring and focal pigmentary retinal mottling,congenital contractures, and marked early hypertonia with symptoms ofextrapyramidal involvement. Furthermore the Zika virus is a neurotropicflavivirus that can potentially cause disease within the central nervoussystem. There is additionally a Worldwide concern over Zika viruscausing Guillain-Barré Syndrome (GBS).

The prevention of the Zika virus disease thus does not only concern thehuman subject being treated but extends to the fetus and newborn in casethe human subject being treated is or will be pregnant. The methodaccording to the invention thus comprises treating the human subject byadministering to the human subject the vaccine or immunogeniccomposition and the treating of the fetus and newborn by administeringto a pregnant human subject or a human subject that intends to becomepregnant or woman of childbearing potential the vaccine or immunogeniccomposition.

Further aspects of the present disclosure relate to methods for usingvaccines and/or or immunogenic compositions described herein containingone or more antigens from at least one Zika virus (e.g., a clonal Zikavirus isolate, a Zika virus comprising a non-human cell adaptationmutation such as a non-human cell adaptation mutation in protein NS1) totreat or prevent Zika virus in a human subject in need thereof and/or toinduce an immune response to Zika virus in a human subject in needthereof.

In certain such methods, the vaccines and/or immunogenic compositionscontains a dose of 1 μg to 15 μg, or 2 μg, or 5 μg, or 10 μg of apurified inactivated whole Zika virus such as a Zika virus with amutation which is a tryptophan to glycine substitution at position 98 ofSEQ ID NO:1 or at a position corresponding to position 98 of SEQ ID NO:1as described herein optionally in combination with one or moreadjuvants, such as 100 μg to about 300 μg or about 150 μg to about 250μg or about 200 μg alum, such as aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition containsa dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purifiedinactivated whole Zika virus comprising a Trp98Gly mutation at position98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQID NO:1, wherein the Zika virus is derived from strain PRVABC59optionally in combination with one or more adjuvants, such as 100 μg toabout 600 μg or about 150 μg to about 250 μg or about 200 μg alum, suchas aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition containsa dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purifiedinactivated whole Zika virus comprising a Trp98Gly mutation at position98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQID NO:1, wherein the Zika virus is derived from strain PRVABC59comprising the genomic sequence according to SEQ ID NO:2 optionally incombination with one or more adjuvants, such as 100 μg to about 600 μgor about 150 μg to about 250 μg or about 200 μg alum, such as aluminumhydroxide.

In certain such methods, the vaccine or immunogenic composition containsa dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purifiedinactivated whole plaque purified Zika virus isolate comprising aTrp98Gly mutation at position 98 of SEQ ID NO: 1, or at a positioncorresponding to position 98 of SEQ ID NO:1, wherein the Zika virus isderived from strain PRVABC59 comprising the genomic sequence accordingto SEQ ID NO:2 optionally in combination with one or more adjuvants,such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about200 μg alum, such as aluminum hydroxide.

In some embodiments, the present disclosure relates to methods fortreating or preventing Zika virus infection in a human subject in needthereof by administering to the human subject a therapeuticallyeffective amount of a vaccine and/or immunogenic composition of thepresent disclosure containing one or more antigens from at least oneZika virus (e.g., a clonal Zika virus isolate, a Zika virus comprising anon-human cell adaptation mutation such as a non-human cell adaptationmutation in protein NS1). In some embodiments, the present disclosurerelates to methods for inducing an immune response to Zika virus in ahuman subject in need thereof by administering to the human subject atherapeutically effective amount of a vaccine and/or immunogeniccomposition of the present disclosure containing one or more antigensfrom at least one Zika virus (e.g., a clonal Zika virus isolate, a Zikavirus comprising a non-human cell adaptation mutation such as anon-human cell adaptation mutation in protein NS1). In some embodiments,the administering step induces a protective immune response against Zikavirus in the human subject. In some embodiments, the human subject ispregnant or intends to become pregnant or woman of childbearingpotential.

The Zika virus vaccines and/or immunogenic compositions disclosed hereinmay be used to protect or treat a human subject susceptible to, orsuffering from a viral infection, by means of administering the vaccineby intranasal, peroral, oral, buccal, sublingual, intramuscular,intraperitoneal, intradermal, transdermal, subdermal, intravaginal,anal, intracranial, intravenous, transcutaneous, or subcutaneousadministration, in particular intramuscular administration. Methods ofsystemic administration of the vaccines and/or immunogenic compositionsof the present disclosure may include conventional syringes and needles,or devices designed for ballistic delivery of solid vaccines (WO99/27961), or needleless pressure liquid jet device (U.S. Pat. Nos.4,596,556; 5,993,412), or transdermal patches (WO 97/48440; WO98/28037). The Zika virus vaccines and/or immunogenic compositions ofthe present disclosure may also be applied to the skin (transdermal ortranscutaneous delivery WO 98/20734; WO 98/28037). The Zika virusvaccines and/or immunogenic compositions of the present disclosuretherefore may include a delivery device for systemic administration,pre-filled with the Zika virus vaccine or immunogenic compositions.Accordingly there is provided methods for treating or preventing Zikavirus infection and/or for inducing an immune response in a humansubject, including the step of administering a vaccine or immunogeniccomposition of the present disclosure and optionally including anadjuvant and/or a carrier, to the human subject, where the vaccine orimmunogenic composition is administered via the parenteral or systemicroute.

The vaccines and/or immunogenic compositions of the present disclosuremay be used to protect or treat a human subject susceptible to, orsuffering from a viral infection, by means of administering the vaccineor immunogenic composition via a mucosal route, such as theoral/alimentary or nasal route. Alternative mucosal routes areintravaginal and intra-rectal. The mucosal route of administration maybe via the nasal route, termed intranasal vaccination. Methods ofintranasal vaccination are well known in the art, including theadministration of a droplet, spray, or dry powdered form of the vaccineinto the nasopharynx of the individual to be immunized Nebulized oraerosolized vaccine formulations are potential forms of the Zika virusvaccines and/or immunogenic compositions disclosed herein. Entericformulations such as gastro resistant capsules and granules for oraladministration, suppositories for rectal or vaginal administration arealso formulations of the vaccines and/or immunogenic compositions of thepresent disclosure.

The Zika virus vaccines and/or immunogenic compositions of the presentdisclosure may also be administered via the oral route. In such casesthe pharmaceutically acceptable excipient may also include alkalinebuffers, or enteric capsules or microgranules. The Zika virus vaccinesand/or immunogenic compositions of the present disclosure may also beadministered by the vaginal route. In such cases, the pharmaceuticallyacceptable excipients may also include emulsifiers, polymers such asCARBOPOL®, and other known stabilizers of vaginal creams andsuppositories. The Zika virus vaccines and/or immunogenic compositionsmay also be administered by the rectal route. In such cases theexcipients may also include waxes and polymers known in the art forforming rectal suppositories.

In some embodiments, the administering step includes one or moreadministrations. Administration can be by a single dose schedule or amultiple dose (prime-boost) schedule. In a multiple dose schedule thevarious doses may be given by the same or different routes e.g. aparenteral prime and mucosal boost, a mucosal prime and parenteralboost, etc. Typically they will be given by the same route, such as byintramuscular or subcutaneous administration. Multiple doses willtypically be administered at least 1 week apart (e.g. about 1 week,about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks,about 11 weeks, about 12 weeks, about 16 weeks, etc., such as 1 to 16weeks apart). Giving two doses separated by from 25-30 days (e.g. 28days, 4 weeks) is particularly useful. In certain such embodiments themode of administration is intramuscular or subcutaneous administration.

The methods of the present disclosure include administration of atherapeutically effective amount or an immunogenic amount of the Zikavirus vaccines and/or immunogenic compositions of the presentdisclosure. A therapeutically effective amount or an immunogenic amountmay be an amount of the vaccines and/or immunogenic compositions of thepresent disclosure that will induce a protective immunological responsein the uninfected, infected or unexposed human subject to which it isadministered. Such a response will generally result in the developmentin the human subject of a secretory, cellular and/or antibody-mediatedimmune response to the vaccine. Usually, such a response includes, butis not limited to one or more of the following effects; the productionof antibodies from any of the immunological classes, such asimmunoglobulins A, D, E, G or M; the proliferation of B and Tlymphocytes; the provision of activation, growth and differentiationsignals to immunological cells; expansion of helper T cell, suppressor Tcell, and/or cytotoxic T cell.

In certain such methods, the vaccines and/or immunogenic compositionscontains a dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purifiedinactivated whole Zika virus such as a Zika virus with a mutation whichis a tryptophan to glycine substitution at position 98 of SEQ ID NO:1 orat a position corresponding to position 98 of SEQ ID NO:1 as describedherein optionally in combination with one or more adjuvants, such as 100μg to about 600 μg or about 150 μg to about 250 μg or about 200 μg alum,such as aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition containsa dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purifiedinactivated whole Zika virus comprising a Trp98Gly mutation at position98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQID NO:1, wherein the Zika virus is derived from strain PRVABC59optionally in combination with one or more adjuvants, such as 100 μg toabout 600 μg or about 150 μg to about 250 μg or about 200 μg alum, suchas aluminum hydroxide.

In certain such methods, the vaccine or immunogenic composition containsa dose of 1 μg to 15 μg, or 2 μg, 5 μg or 10 μg of a purifiedinactivated whole Zika virus comprising a Trp98Gly mutation at position98 of SEQ ID NO: 1, or at a position corresponding to position 98 of SEQID NO:1, wherein the Zika virus is derived from strain PRVABC59comprising the genomic sequence according to SEQ ID NO:2 optionally incombination with one or more adjuvants, such as 100 μg to about 600 μgor about 150 μg to about 250 μg or about 200 μg alum, such as aluminumhydroxide.

In certain such methods, the vaccine or immunogenic composition containsa dose of 1 μg to 15 μg, or 2 μg, or 5 μg or 10 μg of a purifiedinactivated whole plaque purified Zika virus isolate comprising aTrp98Gly mutation at position 98 of SEQ ID NO: 1, or at a positioncorresponding to position 98 of SEQ ID NO:1, wherein the Zika virus isderived from strain PRVABC59 comprising the genomic sequence accordingto SEQ ID NO:2 optionally in combination with one or more adjuvants,such as 100 μg to about 600 μg or about 150 μg to about 250 μg or about200 μg alum, such as aluminum hydroxide.

In certain such methods the administration of the vaccine or immunogeniccomposition induces the generation of neutralizing antibody titers toZika virus in a human subject of greater than 10, or greater than 50, orgreater than 100, or greater than 200 or greater than 1000, or greaterthan 1500, or greater than 2000, or greater than 2000, or greater than3000, as determined by the plaque reduction neutralization test (PRNT)

In certain such methods the administration of the vaccine or immunogeniccomposition induces the generation of neutralizing antibody titers toZika virus in a human subject of greater than 300 or greater than 500,or greater than 1000, or greater than 1500, or greater than 2000, orgreater than 2000, or greater than 3000, or greater than 5000, orgreater than 10,000, as determined by the reporter virus particleneturalization assay (RVP).

In certain such methods the above neutralizing antibody titers areachieved 14 and/or 28 days after the administration.

In certain such methods 14 and/or 28 days after the administration ofthe vaccine or immunogenic composition the generation of neutralizingantibody titers to Zika virus in a human subject is greater than 250, asdetermined by the plaque reduction neutralization test (PRNT)

In certain such methods 14 and/or 28 days after the administration ofthe vaccine or immunogenic composition the generation of neutralizingantibody titers to Zika virus in a human subject is greater than 1000,as determined by the reporter virus particle neturalization assay (RVP).

In certain such methods such titers are achieved 14 and/or 28 days afterthe administration. Such generation of neutralizing antibodies providesfor high seroconversion rates in a Zika virus seronegative population ofat least 20 human subjects. In certain such embodiments theseroconversion rate is at least 60%, at least 70%, at least 80%, atleast 90% at least 95% or 100%.

In certain such methods such titers are achieved 14 and/or 28 days afterthe administration. Such generation of naturalizing antibodies providesfor high seropositvity rates in a population, in particular in a Zikavirus seronegative population, of at least 20 human subjects. In certainsuch embodiments the seropositvity rate is at least 25%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100%.

According to the inventive method the vaccine or immunogenic compositionis administered as a single dose or as multiple doses as e.g. in a first(prime) and a second (boost) administration as e.g. administered 1 to 16weeks apart. In certain such methods the second (boost) administrationis administered at least 28 days after the first (prime) administration.

In certain such methods 14 and/or 28 days after the administration of asingle dose or of multiple doses as e.g. in a first (prime) and after asecond (boost) administration, a high seroconversion rate is achieved ina Zika virus seronegative population, of at least 20 human subjects. Incertain such embodiments the seroconversion rate is at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100%.

In certain such methods including the administration as first (prime)and second (boost) administration 14 and/or 28 days after the second(boost) administration a high seroconversion rate is achieved in a Zikavirus seronegative population, of at least 20 human subjects. In certainsuch embodiments the seroconversion rate is at least 70%, at least 75%,at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100%.

In certain such methods 14 and/or 28 days after the administration assingle dose or after the first (prime) administration a highseroconversion rate is achieved in a Zika virus seronegative population,of at least 20 human subjects. In certain such embodiments theseroconversion rate is at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In certain such methods 14 and/or 28 days after the administration of asingle dose or of multiple doses as e.g. in a first (prime) and after asecond (boost) administration, a high seropositivity rate is achieved ina population, in particular in a Zika virus seronegative population orin a Flavivirus naïve population, of at least 20 human subjects. Incertain such embodiments the seropositvity rate is at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100%.

In certain such methods including the administration as first (prime)and second (boost) administration 14 and/or 28 days after the second(boost) administration a high seropositivity rate is achieved in apopulation, in particular in a Zika virus seronegative population, of atleast 20 human subjects. In certain such embodiments the seropositivityrate is at least 70%, at least 75%, at least 80%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100%.

In certain such methods 14 and/or 28 days after the administration assingle dose or after the first (prime) administration a highseropositivity rate is achieved in a population, in particular in a Zikavirus seronegative population or in a Flavivirus naïve population, of atleast 20 human subjects. In certain such embodiments the seropositivityrate is 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100%.

In certain such methods 14 and/or 28 days after the single dose or primeadministration of the vaccine or immunogenic composition theneutralizing antibody titers to Zika virus in a human subject, inparticular a flavivirus naive human subject, or in particular in a Zikavirus seronegative human subject, are greater than 200, as determined bythe plaque reduction neutralization test (PRNT).

In certain such methods 14 and/or 28 days after the single dose or primeadministration of the vaccine or immunogenic composition the geometricmean neutralizing antibody titers to Zika virus in particular in aflavivirus naive population or in particular in a Zika virusseronegative population of at least 20 human subjects are greater than200, as determined by the plaque reduction neutralization test (PRNT).

In certain such methods 14 and/or 28 days after the single dose or primeadministration of the vaccine or immunogenic composition theneutralizing antibody titers to Zika virus in a human subject, inparticular a flavivirus naive human subject, or in particular in a Zikavirus seronegative human subject, are greater than 1000, as determinedby the reporter virus particle neutralization assay (RVP).

In certain such methods 14 and/or 28 days after the single dose or primeadministration of the vaccine or immunogenic composition the geometricmean neutralizing antibody titers to Zika virus in particular in aflavivirus naive population or in particular in a Zika virusseronegative population of at least 20 human subjects are greater than1000, as determined by the reporter virus particle neutralization assay(RVP).

Such generation of neutralizing antibodies provides for a highseroconversion rates in a flavivirus naive population or in particularin a Zika virus seronegative population of at least 20 human subjectsafter the single dose or prime administration. In certain suchembodiments the seroconversion rate is 25%, at least 30%, at least 40%,at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100%.

Such generation of neutralizing antibodies provides for highseropositvity rates in a population, in particular in a flavivirus naivepopulation or in particular in a Zika virus seronegative population ofat least 20 human subjects after the single dose or primeadministration. In certain such embodiments the seropositvity rate is25%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100%.

In certain methods the administration comprises prime and boostadministration wherein the prime and boost administration takes placefrom about 1 to about 16 weeks apart. In certain such methods the second(boost) administration is administered at least 28 days after the first(prime) administration.

In certain such methods 14 and/or 28 days after the boost administrationof the vaccine or immunogenic composition the neutralizing antibodytiters to Zika virus in a human subject, in particular a flavivirusnaive human subject or in particular in a Zika virus seronegative humansubject, are greater than 1000, or greater than 1500, or greater than3000, as determined by the plaque reduction neutralization test (PRNT).

In certain such methods 14 and/or 28 days after the boost administrationof the vaccine or immunogenic composition the geometric meanneutralizing antibody titers to Zika virus in particular in a flavivirusnaive population or in particular in a Zika virus seronegativepopulation of at least 20 human subjects are greater than 1000, orgreater than 1500, or greater than 3000, as determined by the plaquereduction neutralization test (PRNT).

In certain such methods 14 and/or 28 days after the boost administrationof the vaccine or immunogenic composition the neutralizing antibodytiters to Zika virus in a human subject, in particular a flavivirusnaive human subject or in particular in a Zika virus seronegative humansubject, are greater than 3000, or greater than 5000, or greater than10000, as determined by the reporter virus particle neutralization assay(RVP).

In certain such methods 14 and/or 28 days after the boost administrationof the vaccine or immunogenic composition the geometric meanneutralizing antibody titers to Zika virus in particular in a flavivirusnaive population or in particular in a Zika virus seronegativepopulation of at least 20 human subjects are greater than 3000, orgreater than 5000, or greater than 10000, as determined by the reportervirus particle neutralization assay (RVP).

Such generation of neutralizing antibodies provides for a highseroconversion rates in a flavivirus naive population or in particularin a Zika virus seronegative population of at least 20 human subjectsafter the boost administration. In certain such embodiments theseroconversion rate is at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100%

Such generation of neutralizing antibodies provides for highseropositvity rates in a population, in particular in a flavivirus naivepopulation, of at least 20 human subjects after the boostadministration. In certain such embodiments the seropositvity rate is25%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or 100%

Such generation of neutralizing antibodies provides for a highseroconversion rates in a flavivirus naive population or in particularin a Zika virus seronegative population of at least 20 human subjectsafter the single dose or prime administration. In certain suchembodiments the seroconversion rate is at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100%.

Such generation of neutralizing antibodies provides for a highseroconversion rates in a flavivirus naive population or in particularin a Zika virus seronegative population of at least 20 human subjectsafter the single dose or prime administration. In certain suchembodiments the seropositivity rate is at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100%.

Furthermore, certain aspects of the present disclosure relate to amethod of treating or preventing, in particular preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising antigen froma Zika virus, wherein the vaccine or immunogenic composition isadministered as a single dose or as multiple doses as e.g. in a first(prime) and a second (boost) administration and wherein theadministration of the vaccine or immunogenic composition until 7 daysafter the administration induces systemic side effects in less than 50%of a human subject population of at least 20 human subjects, inparticular in a population of at least 20 flavivirus naïve humansubjects or in particular in a population of at least 20 Zika virusseronegative human subjects.

According to certain aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the administration induces headache symptoms in less than 29% of ahuman subject population of at least 20 flavivirus naïve human subjectsor of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the administration induces fever in 4% or less, and/or fatigue in33% or less, and/or arthralgia in 10% or less, and/or myalgia in 17% orless, and/or malaise in 15% or less of a human subject population of atleast 20 flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces systemic side effects in less than 40% of a humansubject population of at least 20 flavivirus naïve human subjects or ofat least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces

-   -   at least 40% less, or at least 45% less fatigue, and/or    -   no more fever, and/or    -   no more, or at least 10% less, or at least 20% less, or at least        25% less myalgia, and/or    -   no more, or at least 10% less, or at least 20% less malaise        compared to 7 days after the prime administration in a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.

The “%-decrease of adverse events (AEs) after boost administrationcompared to after prime administration” is defined by the followingequation:

${\%\mspace{14mu}{decrease}} = {\frac{\begin{pmatrix}{{\%\mspace{14mu}{subjects}\mspace{14mu}{experiencing}\mspace{14mu}{AEs}\mspace{14mu}{after}\mspace{14mu}{prime}} -} \\{\%\mspace{14mu}{subjects}\mspace{14mu}{experiencing}\mspace{14mu}{AEs}\mspace{14mu}{after}\mspace{14mu}{boost}}\end{pmatrix}}{\%\mspace{14mu}{subjects}\mspace{14mu}{experiencing}\mspace{14mu}{AEs}\mspace{14mu}{after}\mspace{14mu}{prime}} \times 100}$

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces fever in less than 4% or in 0% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or at least 20Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces fatigue in less than 19% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces myalgia in less than 12% or less than 8% of ahuman subject population of at least 20 flavivirus naïve human subjectsor of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces malaise in less than 13% or in 10% or less of ahuman subject population of at least 20 flavivirus naïve human subjectsor of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces headache symptoms in 20% or less, and arthralgiain 8% or less, and fever in less than 4%, and fatigue in less than 19%,and myalgia in less than 12%, and malaise in less than 13% of a humansubject population of at least 20 flavivirus naïve human subjects or ofat least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition comprises a dose of 1 μg to 40 μg of the (one)antigen, wherein the antigen is an inactivated whole virus.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as a single dose administrationor multi dose administration including at least a first (prime) and asecond (boost) administration wherein the vaccine or immunogeniccomposition comprises a dose of about 5 μg of purified inactivated wholevirus.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition until 7 days after the single dose or primeadministration induces systemic side effects in less than 50%, or inless than 45%, or in less than 40% of a human subject population of atleast 20 flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects and/or the administration of the vaccine orimmunogenic composition until 7 days after the boost administrationinduces systemic side effects in less than 40%, or in less than 35%, orin less than 30%, or in less than 25%, or in less than 20%, or in lessthan 15% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces fever in less than3%, or 0%, of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjectsand/or the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces fever in less than4%, or in less than 3%, or 0% of a human subject population of at least20 flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects.

According to certain aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces headache in lessthan 29% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjectsand/or the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces headache in lessthan 20%, or in less than 15%, or in less than 10%, or in less than 5%of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces fatigue in lessthan 30%, or in less than 25%, or in less than 20% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects and/or the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces fatigue in less than 20%, or in less than 15%, orin less than 10% of a human subject population of at least 20 flavivirusnaïve human subjects or of at least 20 Zika virus seronegative humansubjects.

According to certain aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces arthralgia in lessthan 4% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjectsand/or the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces arthralgia in lessthan 5%, or in less than 2%, or in 0% of a human subject population ofat least 20 flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects.

According to certain aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces myalgia in 17% orless of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects and/orthe administration of the vaccine or immunogenic composition until 7days after the boost administration induces myalgia in less than 12%, orin less than 10%, or in less than 5% of a human subject population of atleast 20 flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects.

According to certain aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces malaise in lessthan 10% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjectsand/or the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces malaise in less than14%, or in less than 10%, or in less than 5%, or 0% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces

-   -   at least 70% less, or at least 60% less, or at least 50% less,        or at least 40% less, or at least 35% less, or at least 30% less        systemic side effects, and/or    -   no increase in fever, and/or    -   at least 80% less, or at least 70% less, or at least 60% less,        or at least 50% less, or at least 45% less headache, and/or    -   at least 60% less, or at least 55% less, or at least 50% less,        or at least 45% less, or at least 40% less fatigue, and/or    -   no increase in arthralgia, or at least 80% less, or at least 60%        less, or at least 40% less, or at least 20% less, or at least        10% less arthralgia, and/or    -   no increase in myalgia, or at least 70% less, or at least 60%        less, or at least 40% less, or at least 20% less, or at least        10% less myalgia, and/or    -   no increase in malaise, or at least 80% less, or at least 60%        less, or at least 40% less, or at least 20% less, or at least        10% less malaise compared to 7 days after the prime        administration in a human subject population of at least 20        flavivirus naïve human subjects or of at least 20 Zika virus        seronegative human subjects.

According to certain aspects of the present disclosure the vaccine orimmunogenic composition is administered as a single dose administrationor multi dose administration including at least a first (prime) and asecond (boost) administration wherein the vaccine or immunogeniccomposition comprises a dose of about 10 μg of purified inactivatedwhole virus.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces systemic sideeffects in less than 50% of a human subject population of at least 20flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects and/or the administration of the vaccine orimmunogenic composition until 7 days after the boost administrationinduces systemic side effects in less than 40%, or in less than 35%, orin less than 30% of a human subject population of at least 20 flavivirusnaïve human subjects or of at least 20 Zika virus seronegative humansubjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces fever in less than4% of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects and/orthe administration of the vaccine or immunogenic composition until 7days after the boost administration induces fever in less than 4%, or inless than 3%, or 0% of a human subject population of at least 20flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces headache in lessthan 29%, or in less than 25%, or in less than 20%, or in less than 15%of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects and/orthe administration of the vaccine or immunogenic composition until 7days after the boost administration induces headache in 20% or less of ahuman subject population of at least 20 flavivirus naïve human subjectsor of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces fatigue in 33% orless of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects and/orthe administration of the vaccine or immunogenic composition until 7days after the boost administration induces fatigue in less than 20% ofa human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces arthralgia in 10%or less of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjectsand/or the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces arthralgia in lessthan 5% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces myalgia in 17% orless of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects and/orthe administration of the vaccine or immunogenic composition until 7days after the boost administration induces myalgia in less than 12%, orin less than 10% of a human subject population of at least 20 flavivirusnaïve human subjects or of at least 20 Zika virus seronegative humansubjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces malaise in 15% orless of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects and/orthe administration of the vaccine or immunogenic composition until 7days after the boost administration induces malaise in less than 13%, orin 10% or less of a human subject population of at least 20 flavivirusnaïve human subjects or of at least 20 Zika virus seronegative humansubjects.

According to certain such aspects of the present disclosure the vaccineor immunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces

-   -   at least 40% less, or at least 35% less, or at least 30% less,        or at least 25% less systemic side effects, and/or    -   no increase in fever, or at least 80% less, or at least 60%        less, or at least 40% less, or at least 20% less, or at least        10% less fever, and/or    -   at least 45% less, or at least 40% less fatigue, and/or    -   no increase in arthralgia, or at least 65% less, or at least 60%        less, or at least 40% less, or at least 20% less, or at least        10% less arthralgia, and/or    -   no increase in myalgia, or at least 45% less, or at least 40%        less, or at least 20% less, or at least 10% less myalgia, and/or    -   no increase in malaise, or at least 20% less, or at least 10%        less malaise        compared to 7 days after the prime administration in a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.

According to certain such aspects of the present disclosure the vaccineor immunogenic composition is administered as a single doseadministration or multi dose administration including at least a first(prime) and a second (boost) administration wherein the vaccine orimmunogenic composition comprises a dose of about 2 μg of purifiedinactivated whole virus.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces systemic sideeffects in less than 50%, or in less than 45%, or in less than 40%, orin less than 35% of a human subject population of at least 20 flavivirusnaïve human subjects or of at least 20 Zika virus seronegative humansubjects and/or the administration of the vaccine or immunogeniccomposition until 7 days after the boost administration induces systemicside effects in less than 40%, or in less than 35% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces fever in less than3%, or 0%, of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjectsand/or the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces fever in less than4%, or in less than 3%, or 0% of a human subject population of at least20 flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces headache in lessthan 29%, or in less than 25%, or in less than 20% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects and/or the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces headache in less than 20%, or in less than 15% ofa human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces fatigue in lessthan 30%, or in less than 25% of a human subject population of at least20 flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects and/or the administration of the vaccine orimmunogenic composition until 7 days after the boost administrationinduces fatigue in less than 20%, or in less than 15% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces arthralgia in lessthan 4% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjectsand/or the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces arthralgia in lessthan 8% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces myalgia in 17% orless of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects and/orthe administration of the vaccine or immunogenic composition until 7days after the boost administration induces myalgia in less than 12%, orin less than 10% of a human subject population of at least 20 flavivirusnaïve human subjects or of at least 20 Zika virus seronegative humansubjects.

According to certain such aspects of the present disclosure theadministration of the vaccine or immunogenic composition until 7 daysafter the single dose or prime administration induces malaise in lessthan 10% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjectsand/or the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces malaise in less than13%, or in less than 10%, or in less than 5%, or 0% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects.

According to certain such aspects of the present disclosure the vaccineor immunogenic composition is administered as multiple doses in a first(prime) and a second (boost) administration and the administration ofthe vaccine or immunogenic composition until 7 days after the boostadministration induces

-   -   no increase in fever, and/or    -   at least 50% less, or at least 45% less, or at least 40% less        fatigue, and/or    -   no increase in myalgia, or at least 20% less, or at least 10%        less myalgia, and/or    -   no increase in malaise, or at least 80% less, or at least 60%        less, or at least 40% less, or at least 20% less, or at least        10% less malaise        compared to 7 days after the prime administration in a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.

According to a certain method the human subject or human subjectpopulation is from a Zika endemic region. According to a certain suchmethod the human subject or human subject population is from a Zikaendemic region subject to an outbreak. According to a certain suchmethod the human subject or human subject population is flavivirusnaïve.

According to a certain method the human subject or human subjectpopulation is from a Zika non-endemic region. According to a certainsuch method the human subject or human subject population is from a Zikanon-endemic region travelling to an endemic region. According to acertain such method the human subject or human subject population isflavivirus naïve.

According to a certain method the human subject or human subjectpopulation is Hispanic, Latino, American Indian, Alaska native, Asian,Black or African American, Native Hawaiian or White or a mixturethereof.

According to a certain method the human subject or human subjectpopulation is 18 to 29 years of age.

According to a certain method the human subject or human subjectpopulation is 30 to 49 years of age.

The vaccine or immunogenic composition as disclosed herein is alsodisclosed for use in a method as disclosed herein.

Also disclosed herein is the use of the vaccine or immunogeniccomposition as disclosed herein in the manufacture of a medicament forthe method as disclosed herein.

In some embodiments, the protective immunological response induced inthe human subject after administration of a vaccine and/or immunogeniccomposition containing a non-human cell adapted Zika virus of thepresent disclosure is greater than the immunological response induced ina corresponding human subject administered a vaccine and/or immunogeniccomposition containing a Zika virus that is not adapted for non-humancell growth and/or comprises a different non-human cell adaptationmutation. In some embodiments, the protective immunological responseinduced in the human subject after administration of the vaccine and/orimmunogenic composition containing a non-human cell adapted Zika virusof the present disclosure is at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, or at leastabout 99% greater than the immunological response induced in acorresponding human subject administered a vaccine and/or immunogeniccomposition containing a Zika virus that is not adapted for non-humancell growth and/or comprises a different non-human cell adaptationmutation. Methods of measuring protective immunological responses aregenerally known to one of ordinary skill in the art.

In some embodiments, administration of a vaccine and/or immunogeniccomposition containing a non-human cell adapted Zika virus of thepresent disclosure induces generation of neutralizing antibodies to Zikavirus in the human subject. In some embodiments, administration of avaccine and/or immunogenic composition containing a non-human celladapted Zika virus of the present disclosure induces generation ofneutralizing antibodies to Zika virus in the human subject in an amountthat is greater than the amount of neutralizing antibodies induced in acorresponding human subject administered a vaccine and/or immunogeniccomposition containing a Zika virus that is not adapted for non-humancell growth and/or comprises a different non-human cell adaptationmutation. In some embodiments, administration of a vaccine and/orimmunogenic composition containing a non-human cell adapted Zika virusof the present disclosure induces generation of neutralizing antibodiesto Zika virus in the human subject in an amount that is at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or at least about 99% greater than the than the amount ofneutralizing antibodies induced in a corresponding human subjectadministered a vaccine and/or immunogenic composition containing a Zikavirus that is not adapted for non-human cell growth and/or comprises adifferent non-human cell adaptation mutation. In some embodiments,administration of a vaccine and/or immunogenic composition containing anon-human cell adapted Zika virus of the present disclosure inducesgeneration of neutralizing antibodies to Zika virus in the human subjectin an amount that is at least about 1-fold, at least about 2-fold, atleast about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 6-fold, at least about 7-fold, at least about 8-fold, atleast about 9-fold, at least about 10-fold, at least about 100-fold, orat least about 1000-fold greater than the than the amount ofneutralizing antibodies induced in a corresponding human subjectadministered a vaccine and/or immunogenic composition containing a Zikavirus that is not adapted for non-human cell growth and/or comprises adifferent non-human cell adaptation mutation. Methods of measuringneutralizing antibodies in a human subject are generally known to one ofordinary skill in the art.

Preferably, the therapeutically effective amount or immunogenic amountis sufficient to bring about treatment or prevention of diseasesymptoms. A suitable dosage is about 5 μg or about 10 μg, in particularabout 10 μg,

The present disclosure will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingany aspect or scope of the present disclosure in any way.

EXAMPLES Example 1: Clonal Zika Virus Strain Generation

This example describes the production of Zika virus (ZIKAV) strains witha known research history.

Materials and Methods Vero Cell Maintenance

One vial of WHO Vero 10-87 cells was rapidly thawed in a water bath anddirectly inoculated into 19 mL pre-warmed DMEM (Dulbecco's modifiedminimal essential medium) containing penicillin-streptomycin,L-glutamine 40 mM, and 10% FBS in a T-75 cm² flask at 36° C.+/2° C., at5% CO₂. Cells were allowed to grow to confluency and subcultured usingTryp1E. This flask was expanded to two T-185 cm² flasks, grown toconfluency and subcultured to 31×T-185 cm² flasks and grown until thecells reached 100% confluency. Cells were harvested by trypsinization,centrifuged at 800×g for 10 minutes, and resuspended in DMEM containing10% FBS and 10% DMSO at a concentration of 1.9×10⁷ cells/mL. One vial ofthe Vero cells was rapidly thawed and resuscitated as described aboveinto a T-75 cm² flask. These were subcultured twice to produce a cellbank in 13×T-185 cm² flasks. After trypsinization, the cells werecentrifuged at 800×g and resuspended in freezing media (DMEM containing10% FBS, and 10% DMSO) at a concentration of 4.68×10⁵ cells/mL. Thiscell bank was aliquoted into cryovials.

The Vero cells were grown and maintained in DMEM containingpenicillin-streptomycin, L-glutamine and 10% FBS (cDMEM-10%-FBS).Tryp1Express was used to maintain and trypsinize cells. Two days beforeviral adsorption, 6-well plates were seeded with 4-5×10⁵ cells/well in 3mL of cDMEM-10%-FBS or 7×10⁵ cells in T-25 cm² flasks in 5 mLcDMEM-10%-FBS, or 1×10⁴ cells/well in 96-well plates in 0.1 mLcDMEM-10%-FBS. Incubators were monitored daily to maintain indicatedtemperatures. The Vero cell lines were stored in liquid nitrogen.

Plaque Assay

Viral titers were determined by plaque titration in freshly confluentmonolayers of Vero cells grown in 6-well plates. Frozen aliquots werethawed and ten-fold dilution series of the aliquots were made incDMEM-0%-FBS in 96-well plates. The diluted viruses were maintained onice prior to inoculation of the Vero cell monolayers. At the time ofassay, the growth medium was aspirated from the 6-well plate, and 100 μLof each virus dilution was added to the wells. Virus was adsorbed for 60min at 36° C.±2° C., at 5% CO₂, with frequent (every 10 min) rocking ofthe plates to prevent drying of the cell sheets. Following viraladsorption, 4 mL of a first agarose overlay (1×cDMEM-2%-FBS+0.8%agarose) maintained at 40-41° C. was added to each well. The agarose wasallowed to solidify for 30 min at room temperature, and the plates werethen incubated upside down for 4-6 days at 36° C.+/2° C., at 5% CO₂. TwomL of a second agarose overlay containing 160 μg/mL of neutral red vitaldye was added on day 4. Plaques were visualized on days 5 and 6.

Virus Quantification by TCID50 Assay

Viral titers were also determined by titration in freshly confluentmonolayers of Vero cells grown in 96-well plates. Frozen aliquots werethawed and ten-fold dilution series of the aliquots were made incDMEM-2%-FBS diluent in 96-well plates. The diluted viruses weremaintained on ice prior to inoculation of the Vero cell monolayers. Atthe time of assay, the growth medium was aspirated from the 96-wellplate, and 100 μL of each virus dilution was added to the wells. Theplates were incubated for 5 days at 36° C.+/2° C., at 5% CO₂. The 50%Tissue Culture Infective Dose (TCID50) titer was calculated using theReed/Muench calculator.

Test Articles

Zika virus strain PRVABC59 (one 0.5 mL vial on dry ice) was receivedfrom the Centers for Disease Control and Prevention (CDC) Zika virusidentification was confirmed through RT-PCR. The strain tested negativefor Alphavirus and mycoplasma contamination by PCR. This information issummarized in Table 1.

TABLE 1 PRVABC59 strain information Isolation Patient Strain Informationinformation Prep info Analyses PFU PRVABC59 Human None Passage:Sequencing 6.7 log (Asian) serum; provided Vero(2)C6/36(1) by ion pfu/mLtravel to Prep: Jan. 29, 2016 torrent: gene Puerto Rico Host: C6/36accession in 2015 #KU501215 PFU by plaque assay Identity by RT-PCR (-)For alphaviruses by PCR (-) for mycoplasma by ATCC and ABM PCR

Sequencing

A QIAampViral RNA Mini Spin kit was used to extract RNA from stabilizedvirus harvests of each isolate according to manufacturer protocols.Extracted RNA from each isolate was used to create and amplify 6 cDNAfragments encompassing the entire Zika viral genome Amplified cDNAfragments were analyzed for size and purity on a 1% Agarose/TBE gel andsubsequently gel purified using a Qiagen Quick Gel Extraction Kit. AnABI 3130XL Genetic Analyzer sequencer was used to conduct automaticsequencing reactions. Lasergene SeqMan software was used to analyzesequencing data.

Results

A ZIKAV strain with a known research history that was relevant to thecurrent ZIKAV outbreak in the America's was sought. For this reason,ZIKAV strain PRVABC59 was chosen. To generate a well-characterized virusadapted for growth in Vero cells, the ZIKAV PRVABC59 was first amplifiedin Vero cells (P1).

Flasks of Vero cells (T-175 cm²), 100% confluent, were infected at anMOI of 0.01 in 4 mL of cDMEM-0%-FBS. Virus was adsorbed to the monolayerfor 60 minutes at 36° C.±2° C., at 5% CO₂, then 20 mL of cDMEM-0%-FBSwas applied for viral amplification at 36° C.±2° C., at 5% CO₂. The celllayer was monitored daily for cytopathic effect (CPE) followinginoculation (FIG. 1 ). The supernatant was harvested after 96 hours bycollecting the media and clarifying by centrifugation (600×g, 4° C., 10min). The harvest was stabilized by adding trehalose to a finalconcentration of 18% w/v. The bulk was aliquoted into 0.5 mL cryovialsand stored at −80° C.

The stabilized P1 harvest was analyzed for the presence of infectiousvirus on Vero cell monolayers by a TCID50 assay. Growth kinetics weremonitored by taking daily aliquots beginning on hour 0. Peak titer wasreached by hour 72 (FIG. 2 ).

P1 material was plaque-purified by titrating the harvest from day 3 on6-well monolayers of Vero cells. Plaques were visualized on day 6, and10 plaques to be isolated were identified by drawing a circle around adistinct and separate plaque on the bottom of the plastic plate. Plaqueswere picked by extracting the plug of agarose using a sterile wide borepipette while scraping the bottom of the well and rinsing withcDMEM-10%-FBS. The agarose plug was added to 0.5 mL of cDMEM-10%-FBS,vortexed, labeled as PRVABC59 P2a-j and placed in an incubator overnightat 36° C.±2° C., at 5% CO₂.

Three plaques (PRVABC59 P2a-c) were carried forward for additionalpurification. Each isolate was plated neat in duplicate onto a fresh6-well monolayer of Vero cells. This P2/P3 transition was plaquepurified, and labeled PRVABC59 P3a-j.

Six plaques (PRVABC59 P3a-f) were carried forward for a final round ofpurification. Each isolate was plated neat in duplicate onto a fresh6-well monolayer of Vero cells. This P3/P4 transition was plaquepurified, and labeled PRVABC59 P4a-j.

Six plaques (PRVABC59 P4a-f) from the P4 plaque purification were blindpassaged on monolayers of Vero cells in T-25 cm² flasks. Each plaquepick was diluted in 2 mL cDMEM-0%-FBS—1 mL was adsorbed for 1 hour at36° C.±2° C., at 5% CO₂; the other 1 mL was stabilized with trehalose(18% v/v final) and stored at <−60° C. Following virus adsorption,cDMEM-0%-FBS was added to each flask and allowed to grow at 36° C.±2°C., at 5% CO₂ for 4 days. Virus supernatants were harvested, clarifiedby centrifugation (600×g, 4 C, 10 min), stabilized in 18% trehalose andaliquoted and stored at <−60° C. This P5 seed was tested by TCID50 forZika virus potency (FIG. 3 ).

Confluent monolayers of T-175 cm² flasks of Vero cells were infectedwith each of the six clones of PRVABC59 (P5a-f) at an MOI of 0.01 in 4mL cDMEM-0%-FBS. The virus was allowed to adsorb for 60 minutes at 36°C.+/2° C., at 5% CO₂, after which 20 mL of cDMEM-0%-FBS was added toeach flask and allowed to grow at 36° C.+/2° C., at 5% CO₂. Vero cellmonolayer health and CPE was monitored daily. Virus was harvested ondays 3 and 5 as indicated (FIG. 4 ). The P6 strain harvests from days 3and 5 were pooled, stabilized with 18% trehalose, aliquoted and stored<−60° C.

Each of the six clones of PRVABC59 (P6a-f) were tested for Zika virus invitro potency (FIG. 5 ). The potency was determined by two differentmethods, TCID50 and plaque titration. The TCID50 was calculated byvisual inspection of CPE (microscope) and by measuring the difference inabsorbance (A560-A420) of the wells displaying CPE (yellow in color)compared with red (no CPE). The plates were read on a plate reader, andapplied to the same calculator as the microscopically read-plates(absorbance). The values in TCID50 between the two scoring techniquesare quite similar, while the values obtained by plaque titration arelower.

A summary of the generation of the P6 virus and characterization isshown in Table 2 below.

TABLE 2 Summary of virus passage and characterization for the generationof clonal ZIKAV strains Passage Seed production/purificationCharacterization P1 Virus amplification in Vero TCID50 titer P2 AmplifyP1 by plaque titration plaque purification P3 Pick and passage plaquesfrom P2 plaque plaque purification assay P4 Pick and passage plaquesfrom P3 plaque plaque purification assay P5 Amplify P4 plaques (a-f)TCID50 titer P6 Amplify P5 (a-f) virus TCID50 titer, plaque phenotype,genotype, growth kinetics

An isolated Zika virus clone that closely resembled the envelopeglycoprotein sequence of the original isolate was sought, since theenvelope protein of flaviviruses is the dominant immunogenic portion ofthe virus. PRVABC59 clones P6a, P6c, P6d and P6f contained a G→Tmutation at nucleotide 990 in the envelope region (G990T), resulting inan amino acid mutation of Val→Leu at envelope residue 330, whereas theenvelope gene of PRVABC59 clones P6b and P6e were identical relative tothe reference strain (GenBank ref KU501215.1) (Table 3 and FIG. 6 ).

TABLE 3 Sequencing of PRVABC59 P6 clones Envelope sequencing (referencegene from PRVABC59; accession #KU501215) Strain Nucleotide Amino AcidMutation Comments PRVABC59 P6a Env-990: Env-330: Val/Leu Mutation in 3of 4 G→T Val330→*Leu reads. PRVABC59 P6b Env-1404: Wild type Wild typeWild type relative to T→G silent reference. PRVABC59 P6c Env-990:Env-330: Val/Leu Mutation in 3 of 4 G→T Val330→*Leu reads. PRVABC59 P6dEnv-990: Env-330: Val/Leu Mutation in 2 of 2 G→T Val330→*Leu reads.PRVABC59 P6e Wild type Wild type Wild type Wild type relative toreference. PRVABC59 P6f Env-990: Env-330: Val/Leu Mutation in 2 of 2reads. G→T Val330→Leu 190 bp not sequenced (aa 421-484). PRVABC59 P6bEnv-1404 Wild-type Silent Mutation in 2 of 2 reads T→G NS1-292 NS1-98Trp/Gly Mutation in 2 of 2 reads T→G Trp98→*Gly PRVABC59 P6e NS1-292NS1-98 Trp/Gly Mutation in 2 of 2 reads T→G Trp98→*Gly

The two clones lacking mutations in the envelope sequence were thensubjected to full genome sequencing. Sequencing results are summarizedin Table 3 above. Sequence analysis revealed a T→G substitution atnucleotide 292 in the NS1 region for both clones, resulting in a Trp→Glymutation at NS1 residue 98. This mutation was also later confirmedthrough deep sequencing. The NS1 W98G mutation is located in theintertwined loop of the wing domain of ZIKAV NS1, which has beenimplicated in membrane association, interaction with envelope proteinand potentially hexameric NS1 formation. While other tryptophan residues(W115, W118), are highly conserved across flaviviruses, W98 is not (FIG.7 ). Interestingly, however, 100% conservation of the W98 residue isobserved across 11 different ZIKAV strains, including those from theAfrican and Asian lineages. The identified mutations in each strain aresummarized in Table 4.

TABLE 4 Summary of mutations identified in PRVABC59 P6 clones CloneNucleotide Amino Acid Mutations identified in envelope P6a G990T V330LP6b T1404G (silent) P6c G990T V330L P6d G990T V330L P6e none none P6fG990T V330L Additional mutations identified in genome P6b NS1-T292GNS1-W98G P6e NS1-T292G NS1-W98G Ref sequence: KU501215.1 (PRVABC59)

Phenotypic analysis of the ZIKAV PRVABC59 P6 stocks was conducted tocharacterize the ZIKAV clones. As illustrated in FIG. 8 and quantifiedin FIG. 9 , each clonal isolate consisted of a relatively homogeneouspopulation of large-sized plaques as compared to the P1 virus, which hada mixed population of large and small plaques. These data suggest thesuccessful isolation of single ZIKAV clones.

Next, growth kinetics analyses in Vero cells of the ZIKAV PRVABC59 P6clones were analyzed. Vero cells were infected with 0.01 TCID50/cell ofeach ZIKAV P6 clones in serum free growth medium. Viral supernatantsamples were taken daily and simultaneously assayed for infectious titerby TCID50 assay. For all P6 clones, peak titer occurred between day 3and 4 (˜9.0 log₁₀ TCID50/mL). There was no significant difference ingrowth kinetics of the various P6 clones (FIG. 10 ).

Taken together, the results indicate that a Zika virus seed wassuccessfully generated. This seed selection required understanding ofgrowth history, kinetics, yield, genotype, and phenotype of the virus.Importantly, clonal isolation of the Zika virus strains allowed for thesuccessful purification of the virus away from contaminating agents(e.g., adventitious agents that may be in the parental human isolate).Interestingly, three sequential plaque purifications succeeded inquickly selecting Vero-cell adapted virus (strains P6a-f), where thesestrains were able to replicate well in serum-free Vero cell cultures,with strain P6a, c, d, and f harboring a mutation in the viral envelopeprotein, while strains p6b and p6e obtained a mutation in the viral NS1protein (with no modification to the viral envelope). Additionally, theVero-adapted strains enabled efficient and reproducible growth andmanufacture of subsequent viral passages propagated from these strains.Without wishing to be bound by theory, the Env-V330L mutation observedin strains P6a, c, d, and f may potentially be a result of in vitroadaptation, as a mutation at Env 330 was also observed upon passaging inVero cells (Weger-Lucarelli et al. 2017. Journal of Virology). Becausethe envelope protein is the dominant immunogenic epitope of Zika virus,strains containing a Vero adaptive mutation in Env may negatively impactvaccine immunogenicity. Without wishing to be bound by theory, theadaptation mutation in protein NS1 appears not only to enhance viralreplication, but may also reduce or otherwise inhibit the occurrence ofundesirable mutations, such as in the envelope protein E (Env) of theZika virus. In addition, NS1 may be known to bind to the Envelopeprotein during the life cycle of the virus. This mutation (NS1 W98G) maybe implicated in changing the ability of the NS1 to associate, andpossibly co-purify, with the virus during downstream processing. NS1 isalso known to be immunogenic, and could be implicated in the immuneresponse to the vaccine.

Example 2: Preclinical Immunogenicity and Efficacy of a PurifiedInactivated Zika Virus Vaccine (PIZV) Derived from the P6b and P6eStrains

The following example describes the preclinical immunogenicity andefficacy in CD1 and AG129 mice of an inactivated Zika virus vaccine(PIZV) derived from the P6b and P6e strains. As described in Example 1,six clones were generated from the epidemically relevant PRVABC59strain, and two (P6b and P6e) were chosen for further preclinicalimmunogenicity and efficacy studies.

Materials and Methods Purification, Inactivation and Formulation of aZika Virus Vaccine

A lot of inactivated ZIKAV vaccine, suitable for use in preclinicalimmunogenicity and efficacy studies, was generated and characterized.Virus was amplified from the P6b and P6e strains by infecting flasks ofconfluent Vero cells at a MOI of 0.01. Virus was adsorbed for 1 hour at36° C.±2° C./5% CO₂. Following adsorption, 20 mL of cDMEM-0%-FBS wasadded to each flask, and incubated at 36° C.±2° C./5% CO₂ for five days.Cell supernatants were harvested on day 3 and 5 post-infection, and celldebris was clarified by centrifugation.

For each isolate, clarified supernatants were pooled, stabilized in DMEMcontaining 18% trehalose and stored at <−60° C. Pooled, clarified virussupernatants were thawed in a 37° C. water bath and treated withbenzonase overnight at 4° C. Following benzonase treatment, each samplewas applied to a Sartorius PP3 depth filter. Following depth filtration,each sample was applied to a Centricon Plus-70 tangential flowfiltration (TFF) device. Retentate was buffer exchanged, diluted, andapplied to a Sartorius SartobindQ IEXNano. Each sample was applied to asecond Sartorius SartobindQ IEXNano and eluted using a 3 step-elutionprocess with 250 mM, 500 mM, and 750 mM NaCl. Following MonoQchromatography and dilution, each 250 mM eluate was applied to aCentricon Plus-70 cross flow filtration (CFF) device for bufferexchange, diluted to 35 mL with PBS, and stored at 2-8° C.

For formalin inactivation, freshly prepared 1% formaldehyde was addeddropwise to each purified sample with gentle swirling to obtain a finalformaldehyde concentration of 0.02%. Samples were incubated at roomtemperature (˜22° C.) for 14 days with daily inversion. Formaldehyde wasneutralized with sodium metabisulfite for 15′ at room temperature beforebeing applied to a Centricon Plus-70 tangential flow filtration (TFF)device. Buffer exchange was performed four times by the addition of 50mL Drug Substance Buffer (10 mM NaH₂PO₄, 50 mM NaCl, 6% sucrose, pH7.4). Each sample was then diluted to 15 mL with Drug Substance Buffer,sterilized using a 0.2 m syringe filter, aliquoted into sterilestoppered glass vials (0.5 mL per vial) and frozen at <−60° C.

Virus inactivation was confirmed by TCID50 assay and double infectivityassay. Briefly drug substance sample was applied to C6/36 cells andallowed to amplify for 6 days. Supernatant from C6/36 cells was appliedto Vero cells and CPE was monitored for 8 days. For drug productformulation, vials of PIZV drug substance were thawed, pooled accordingto sample type, and diluted to 1 μg/mL or 10 μg/mL in PBS with orwithout Alhydrogel (Brenntag; 0.5 mg/mL final, 0.050 mg/dose) andincubated overnight at 2-8° C. with gentle agitation. The resulting drugproduct lots were then aliquoted into sterile stoppered glass vials andstored at 2-8° C. until use. FIG. 11 provides a summary of the stepsused to prepare drug product.

Mouse Immunization and Challenge

For the immunogenicity study, six-week old male and female Swiss-ICR(CD-1) mice were divided into 6 groups (n=10/group). On Day 0, mice ingroups 1-5 were inoculated with 0.1 mL of vaccine by the intramuscular(i.m.) route (2×0.05 mL injections). Mice in group 6 were inoculatedwith PBS as a placebo control. Mice were boosted on day 28 and 56 usingthe same dosage and vaccine type as day 0. Blood samples were collectedon day −1 (pre-immune), day 27 (prime), day 42 (boost 1) and day 70(boost 2).

For the immunogenicity and efficacy study, four-week old male and femaleAG129 mice were divided into 7 groups (n=5/group). On Day 0, mice ingroups 1-6 were inoculated with 0.1 mL of vaccine by the intramuscular(i.m.) route (2×0.05 mL injections). Mice in group 7 were inoculatedwith PBS as a placebo control. Mice were boosted on day 28 using thesame dosage and vaccine type as on day 0. Blood samples were collectedfrom the tail vein on day −1 (pre-immune), day 27 (prime) and day 55(boost). At the time of euthanization, mice were bled via cardiacpuncture under deep anesthesia with isofluorane (terminal). On day 56,mice were intraperitoneally challenged with 10⁴ plaque forming units(PFU) of ZIKAV PRVABC59.

Serum Transfer

Serum was collected from PIZV-vaccinated and challenged AG129 mice, andwere frozen after pooling (groups 1, 2, 4, and 5 of Table 6). The serumpool was thawed, and the test articles were generated by three-folddilutions of the serum pool in PBS. A placebo was generated using 3-folddilutions of AG129 normal mouse serum in PBS.

The test articles were administered as 0.1 mL intraperitoneal injectionsinto AG129 mice (an equivalent volume of the placebo article wasadministered to control mice). Animals were then challengedintraperitoneally with 10⁴ plaque forming units of Zika virus strainPRVABC59 in 100 μL.

Allowable blood volume by weight was collected as whole blood by tailbleeding from ten mice on day −11 (pre-immunization). Whole blood wascollected from each mouse on day 1 (primary, circulating Nab) and day 4(viremia) by tail bleeding. Terminal bleeding after lethal challenge wasperformed by heart puncture under deep anesthesia for larger volumebefore euthanization by cervical dislocation. Blood samples werecollected in microtainer SST serum separation gel tubes and allowed toclot for at least 30 min before separation of serum by centrifugation(10,000×g for 2 min) and frozen at −80° C.

Plaque Reduction Neutralization Test

Neutralizing antibody titers were determined by a plaque reductionneutralization test (PRNT) as described previously (See e.g., Osorio etal. Lancet Infect Dis. 2014 September; 14(9):830-8).

Reporter Virus Particle (RVP) Neutralization Assay

Neutralizing antibody titers were analyzed by titration of serum sampleswith a constant amount of Zika RVPs in Vero cells grown in 96-wellplates. RVPs contained the prME proteins of Zika (strain SPH2012) and aDengue-based Renilla luciferase reporter. Briefly, sera were heatinactivated at 56° C. for 30 min, diluted, and then incubated at 37° C.with RVPs. The serum/RVP mixture was then mixed with Vero cells andincubated for 72 hours at 37° C.±2° C./5% CO₂ before detection withluciferase substrate. Data was analyzed using JMP11 non-linear 4parameter analysis, normalized to a positive tracking control andeffective dose 50% (EC50) was reported.

Unless indicated to the contrary, all additional experimental methodswere carried out as described in Example 1 above.

Results

To assess the immunogenicity of the PIZV candidates in 6 week old maleand female CD-1 mice, groups of CD-1 mice (N=10/group) were immunized bythe i.m. route with either a 0.1 μg (+alum), 1.0 μg (+alum) dose of avaccine derived from either ZIKAV PRVABC69 P6b or P6e virus strains. Toassess the need for adjuvant, a group of animals was vaccinated with 0.1μg of vaccine derived from P6e and lacking alum adjuvant. Vaccinationsoccurred on days 0, 28, and 56, with group 6 receiving PBS as a placebocontrol (FIG. 12A and Table 5).

TABLE 5 PIZV formulations and challenges in CD-1 mice Group Strain Dose(μg) Alum (μg) N 1 P6b 0.1 0.50 10 2 P6b 1.0 0.50 10 3 P6e 0.1 0.50 10 4P6e 1.0 0.50 10 5 P6e 0.1 — 10 6 Placebo (PBS) — — 10

Following vaccination, serum samples collected after primary (day 27),secondary (day 40) and tertiary (day 70) immunizations were tested forZIKAV-specific neutralizing antibodies by RVP neutralization assay (FIG.12B). Twenty-seven days after receiving the first dose, a slightneutralizing antibody response was observed in mice vaccinated with PIZVderived from either clone containing alum, as compared to the PBSplacebo control group. Importantly, this response increasedsignificantly upon a second immunization (day 40), but was notadditionally enhanced upon immunization with a third dose (day 70). Noneutralizing antibody response was observed in mice vaccinated withnon-adjuvanted vaccine (FIG. 12B).

To assess the immunogenicity and protective efficacy of the PIZVcandidates, groups of 4 week old AG129 mice (n=5/group) were immunizedby the i.m. route with either a 0.1 μg dose (+alum), 1.0 μg dose (+alum)or 0.1 μg dose (−alum) of a vaccine derived from either the ZIKAVPRVABC59 P6b or P6e stocks on days 1 and 28 (FIG. 13A and Table 6).

TABLE 6 PIZV formulations and challenges in AG129 mice Group Sex StrainDose (μg) Alum (μg) N 1 F P6b 0.1 0.50 5 2 F P6b 1.0 0.50 5 3 F P6b 0.1— 5 4 M P6e 0.1 0.50 5 5 M P6e 1.0 0.50 5 6 M P6e 0.1 — 5 7 M Placebo(PBS) — — 5

Following vaccination, vaccinated and control mice wereintraperitoneally challenged at day 56 with 10⁴ PFU of ZIKAV PRVABC59(low passage). Serum samples collected after primary (D27) and secondary(D55) immunizations were tested for ZIKAV-specific neutralizing antibodyresponse (FIG. 13B and Table 7). Only groups receiving the high dose ofalum-adjuvanted vaccine (groups 2 and 5) elicited a neutralizingantibody response after a single immunization, which increaseddramatically after boosting. In contrast, groups receiving either thelow or high dose of alum-adjuvanted vaccine produced a high neutralizingantibody response after a second dose. Upon receiving two doses ofvaccine, there was no statistical difference between groups of micereceiving alum-adjuvanted vaccine, regardless of the dosage or thederivation from the P6 clone.

TABLE 7 ZIKAV-specific neutralizing antibody response Serum neutralizingantibody titers D27 (prime) D55 (boost) Group Formulation GMT % sc GMT %sc 1 P6b 0.1 μg + <20 40 1280 100 alum 2 P6b 1.0 μg + 135 80 2229 100alum 3 P6b 0.1 μg − <20 0 <20 0 alum 4 P6e 0.1 μg + <20 20 640 100 alum5 P6e 1.0 μg + 30 100 905 100 alum 6 P6e 0.1 μg − <20 0 <20 20 alum 7PBS <20 0 <20 0

All groups were also monitored for mortality, morbidity and weight lossfor 21 days post challenge. Viremia following challenge was detected andquantitated by plaque titration. Mice vaccinated with a low or high doseof PIZV candidates formulated with alum (groups 1, 2, 4 and 5) werefully protected from lethal ZIKAV challenge, as assessed by the plaquereduction neutralization test (PRNT) assay, as well as a comparablesecondary neutralization assay (Table 8). No weight loss or clinicalsigns of illness were observed in vaccinated mice, none had detectableinfectious viremia three days post challenge, and all mice vaccinatedwith either low or high dose antigen+alum adjuvant survived to 21 dayspost-challenge (FIGS. 14-16 ). In contrast, challenge of all naïve miceresulted in high viremia on day 2 post challenge and morbidity/mortalitybetween day 10 and 18 post challenge (median survival=D13).Additionally, challenge of mice vaccinated with a non-alum-adjuvantedlow dose vaccine derived from strain P6b resulted in high viremia on day2 post challenge and a median survival day similar to the placebocontrol group, while mice vaccinated with a non-alum-adjuvanted low dosederived from clone e remained partially protected with a median survivalof 19 days. These results indicate immunization is more effective withalum, secondary immunization may be a requirement, and that low dose wasas effective as high dose.

TABLE 8 Serum neutralizing antibody titers Serum neutralizing antibodytiters Terminal (post challenge) Pool PRNT₅₀ Secondary assay Alum (1, 2,4, 5) 10240 20480 No alum (3, 6) 2560 2560 PBS (7) 1280 1280

Additionally, the presence of NS1 in the vaccine drug substance (DS)produced from whole inactivated P7b and P7e virus (one additionalpassage from the P6b and P6e strains, respectively) was tested. Asandwich ELISA was performed using plates pre-coated with a monoclonalantibody reactive to both Asian and African lineages of Zika virus NS1,but non-cross-reactive to Dengue NS1. Duplicate 2-, 4-, 8-, 16-, and32-fold dilutions of DS were prepared, and were compared to a standardcurve using recombinant purified NS1 in duplicate at a concentration of0-8 ng/mL. Duplicate dilutions of DS buffer alone were prepared asnegative controls. Bound NS1 was detected with anti-NS1 HRP-conjugate,and absorbance (A450-A630) of the wells with DS buffer alone wassubtracted from the absorbance measured in the wells containing thematching DS samples. Results of the sandwich ELISA are shown in Table 9below. Interestingly, NS1 was observed to co-purify with the vaccinedrug substance preparations, suggesting that viral NS1 may be animmunogenic component of the whole inactivated virus vaccine.

TABLE 9 NS1 ELISA Strain in Predicted Predicted vaccine Sample log StdLower Upper Dilution concentration preparation OD ng/mL Error 95% 95%Factor (ng/mL) P7b 3.61 0.951 0.018 0.915 0.986 32 ~285 P7e 3.79 0.9800.023 0.935 1.024 32 ~306

The threshold of neutralizing antibody (Nab) needed to confer protectionfrom wild-type Zika virus challenge after passive transfer of antibodieswas next tested. (Tables 10A and B).

TABLE 10A design of passive transfer study in AG129 mice Group TestArticle Serum dilution Predicted Nab titer before IP 1 100 μL 1/36827/3.83 2 100 μL 1/9 2276/3.36 3 100 μL  1/27  759/2.88 4 100 μL  1/81 253/2.40 5 100 μL  1/243  84/1.93 6 100 μL  1/729  28/1.45 7 100 μL  1/2187   9/0.97 8 100 μL PBS —

TABLE 10B Timing of passive transfer study in AG129 mice DescriptionStudy Day Passive transfer Day 0 Primary Bleed Day 1 (AM) Challenge (PM)Day 1 Viremia Bleed Day 4 Terminal Bleed Day 29 for survivors

Pooled serum from vaccinated and challenged AG129 mice was seriallydiluted 3-fold in PBS and intraperitoneally injected into 7 groups(N=5/group) of 5-6 week old AG129 mice. Pre-immune AG129 mouse serum wasused as placebo control (group 8). Following passive transfer (˜16-19hours later), whole blood was collected and serum was separated bycentrifugation from each mouse prior to virus challenge fordetermination of circulating neutralizing antibody titer (FIG. 17 ).Just prior to virus challenge, groups of mice (designated groups 1, 2,3, 4, 5, 6, 7, 8) had mean log₁₀ neutralizing antibody titers of 2.69,2.26, 1.72, 1.30, <1.30, <1.30, <1.30, <1.30, respectively.

Twenty four hours following passive transfer of ZIKV nAbs, mice wereintraperitoneally challenged with 10⁴ pfu of ZIKV PRVABC59. Followingchallenge, animals were weighed daily and monitored 1-3 times a day for28 days for signs of illness. A clinical score was given to each animalbased on the symptoms (Table 11). Animals that were moribund and/orshowed clear neurological signs (clinical score ≥2) were humanelyeuthanized and counted as non-survivors.

TABLE 11 Description of clinical scores given while monitoring formorbidity and mortality Score Description 0 Normal appearance andbehavior 1 Slightly ruffled fur and/or general loss of condition 2Increases in above behavior/appearance, breathing changes, twitching,anti-social behavior 3 First signs of neuropathy-Severely hunchedposture, partial paralysis (immobility, unsteady gait, flaccid hindlegs, severe twitching), or full paralysis 4 Found dead without showingsigns of score of 2 or 3 first

Signs of disease began appearing nine days after challenge in thecontrol group (group 8) and groups 5-7, with a corresponding loss inweight (FIG. 18 ). Whole blood was collected and serum was separated bycentrifugation from each animal three days post challenge. Serum sampleswere analyzed for the presence of infectious ZIKV using a plaquetitration assay (FIG. 19 ). The mean infectious titer (log 10 pfu/mL)for mice in groups 1-8 were: 1.66, 2.74, 4.70, 4.92, 7.24, 7.54, 7.54and 7.46, respectively. Importantly, mice in groups 1-4 with detectablelevels of ZIKV neutralizing antibodies (≥1.30 log₁₀) had statisticallysignificant lower levels (102.5- to 106.0-fold lower titers) of viremia(p=0.0001, 0.0003, 0.0007 and 0.0374) than control mice. These resultssuggested that detectable levels of ZIKV neutralizing antibodies (≥1.30log₁₀) reduced viremia in a dose-dependent manner

The median survival day of mice in groups 1-8 were: not determined, day17, day 17, day 13, day 11, day 11, day 11, and day 10, respectively(FIG. 20 ). Importantly, the survival curves for groups of mice withdetectable ZIKV neutralizing antibody titers (groups 1-4) werestatistically different compared to the control group (group 8)(p=0.0019, 0.0019, 0.0019, 0.0153, respectively). These resultssuggested that detectable levels (≥1.30 log₁₀) of ZIKV neutralizingantibodies delayed onset of disease in a dose-dependent manner

Finally, the ZIKV neutralizing antibody titer of each animal was graphedagainst its corresponding viremia titer and linear regression analysiswas performed. A highly inversely correlated relationship between ZIKVneutralizing antibody titers and viremia levels at day 3 post-challengewas observed (FIG. 21 ). A summary of the results from the passivetransfer studies is shown in Table 12 below.

TABLE 12 Summary of passive transfer results Circulating Viremia ZIKV(D3) % Median Serum nAb log10 survival survival Group dilution GMTpfu/mL (D28) day 1 1/3 2.69 ± 0.17 1.66 ± 0.62 20 24 2 1/9 2.26 ± 0.132.73 ± 0.68 0 17 3 127 1.72 ± 0.16 4.69 ± 0.77 0 17 4 1/81 1.30 ± 0.164.94 ± 1.29 0 13 5 1/243 <1.30 7.25 ± 0.10 0 11 6 1/729 <1.30 7.54 ±0.31 0 11 7 1/2187 <1.30 7.52 ± 0.39 0 11 8 PBS <1.30 7.47 ± 0.37 0 10

While no groups of mice receiving ZIKAV neutralizing antibodies werefully protected from lethal ZIKAV challenge in this experiment, reducedviremia levels and delayed onset of disease in a dose-dependent manneramong the groups of mice with detectable levels of circulating ZIKAVneutralizing antibody titers was demonstrated.

Taken together, preclinical data from both CD-1 and AG129 mouse studiesindicate that a PIZV derived from separate and well-characterized viralclones are immunogenic and able to provide protection against challengewith wild-type ZIKAV. Importantly, a low and high vaccine dose eliciteda similar neutralizing antibody response after two doses, and providedsimilar levels of protection against lethal ZIKAV challenge.Interestingly, mice vaccinated with an unadjuvanted PIZV candidate alsoshowed partial protection from ZIKAV challenge. Vaccine antiserasignificantly diminished viremia in passively immunized AG129 mice, andprolonged survival against lethal ZIKAV challenge. These results alsodemonstrate that the well-characterized PIZV candidates were highlyefficacious against ZIKAV infection in the highly ZIKAV-susceptibleAG129 mouse model.

Additionally, it was found that the sequence of a PRVABC59 (fromPRVABC59 P6e) at passage 7 was genetically identical to that of passage6. This was surprising given that flaviviruses are generally regarded asgenetically labile. PRVABC59 P6e was selected as the master virus seeddue in part to its genetic stability over 7 passages. Without wishing tobe bound by theory, it is believed that this enhanced genetic stabilitymay be due to the single amino acid substitution (W98G) in the wingdomain of NS1, as this was the only mutation observed in the Verocell-adapted PRVABC59 P6 genome. Additionally, genetic stability andhomogeneity is advantageous in that it reduces variability and increasesreproducible production of subsequent strains that may be used forvaccine formulation.

Example 3: Preclinical Assessment of the Phenotype of the P6a and P6eStrains Materials and Methods

AG129 mice (lacking interferon α/β and γ receptors) are susceptible toZIKV infection and disease, including severe pathologies in the brain.14-week-old AG129 mice were intraperitoneally infected with 10⁴ and 10³pfu of the ZIKV passage 6 clones a (P6a) and e (P6e).

Mice were weighed and monitored daily (up to 28 days) for clinical signsof illness (weight loss, ruffled fur, hunched posture, lethargy, limbweakness, partial/full paralysis). Additionally, analysis of viremia wasperformed by plaque titration of serum samples collected three dayspost-challenge as described in Example 1.

Results

Infection with P6e resulted in 100% mortality (median survival time=12.5days), while infection with P6a resulted in only 33% mortality (mediansurvival time=undetermined) (FIG. 22 ). In agreement with this, preMVSP6e infected mice showed greater weight loss as compared to PRVABC59 P6ainfected mice (3). No statistical difference was found in mean groupviremia levels between groups of mice infected with PRVABC59 P6a or P6e(FIG. 24 ). These data suggest that growth kinetics alone may not be akey determinant (since both strains produced similar viremia, andsimilar peak titers in vitro) and that a characteristic of the Envelopeprotein could be important for virulence (of a wildtype strain) andimmunogenicity (of an inactivated candidate).

Example 4: Completeness of Inactivation Assay to Determine Effectivenessof Inactivation

A double-infectivity assay also called completeness of inactivation(COI) assay was developed to determine the effectiveness offormalin-inactivation (0.01% formaldehyde) and potential residualinfectious viral activity of purified inactivated zika virus (PIZV) bulkdrug substance (BDS).

Sample preparation: Four Purified Inactivated Zika Vaccine (PIZV) lots(Tox lots 1-4) of clone e as described above were manufactured by growthin Vero cells. Supernatants from 4 daily harvests (totaling about 4000mL) were purified by chromatography followed by addition of formaldehydeto a final concentration of 0.01%. w/v Inactivation was allowed toproceed for 10 days at 22° C. In Process Control (IPC) samples wereremoved on a daily basis from the bulk drug substance (BDS) duringinactivation for characterization and analytics. The daily IPC sampleswere neutralized with sodium metabisulfite and dialysed into DMEM (viralgrowth media). The samples contain the purified inactivated Zika virus.On the final day of inactivation, the remaining volume of BDS sampleswas not neutralized, but was processed with TFF to remove formaldehydeand buffer exchanged into PBS.

Completeness of inactivation assay (COI): The COI assay was used foranalysis of the effectiveness of inactivation in the daily IPC samplesto understand the kinetics of inactivation, and the final BDS. Formaximum sensitivity, two cell lines, Vero and C6/36, were initiallyutilized in this assay to detect potential live virus in the IPC and DSsamples. When Zika virus infects Vero cells in the presence of growthmedium containing phenol red, the by-products of cell death cause a dropin pH. Consequently, the media color changes from red/pink to yellow,indicative of this acidic shift in the media pH. This phenomenon iscaused by the apoptosis and cytopathic effects (CPE), which refers tothe observed changes in the cell structure of host cells that are causedby viral invasion, infection, and budding from the cells during viralreplication. Ultimately, while both C6/36 mosquito and Vero cells are apermissive cell line for infection, Zika virus infection kills only Verocells in vitro. Therefore, Vero cells were used as the indicator cellline for the assay. In contrast, C6/36 cells which are derived from anatural host vector for Zika virus do not exhibit a CPE upon Zikainfection and do not lyse. The media does not change color and theviability of the C6/36 cells is not altered.

The assay is thus split in two parts: The first part of the assay allowsfor parallel amplification of potentially live viral particles on96-well plates of the two susceptible cell lines for six days. Thesecond step of the assay involves the transfer of the supernatant of the96-well plates (including potentially amplified particles) onto 6-wellplates containing monolayers of Vero cells, and incubation for another 8days to allow for viral infection and a cytopathic effect to develop onthe Vero cells. Any CPE observed was confirmed using a light microscope.

Although described in detail with respect to the use of 96 well platesin the first part of the assay, i.e. the culture in C6/36 cells, and sixwell plates in the second part of the assay, i.e. the culture of Verocells to observe a cytopathic effect, the assay can be easily scaled upaccording to the following table:

Assay part 2: transfer to Vero (must accommodate Assay part 1: BDSapplication (must fall within pooled recommended vol range) volume for #vessels # vessels transfer) vol required required pooled vol Recommendedinoculum for 15X for 15X volume transferred Surface volume mL per wellscale-up; scale-up; for mL inoculum plate or area range (for sample (orper 2-fold 5-fold transfer sample per well flask (cm²) growth) per cm²flask) dilution dilution (mL) per cm² (or flask) 96-well 0.32 100-200 uL0.3125 0.1 format 12-well 3.8 0.076-1.14 ml 0.3125 1.188 6.48 16.2111.88 format 6-well 9.5 1.9-2.9 mL 0.3125 2.969 4.32 10.81 17.81 0.05260.1 format T25 flask 25 5-7.5 mL 0.3125 7.813 9.86 24.64 7.813 0.05261.32 format T75 flask 75 15-22.5 mL 0.3125 23.438 3.29 8.21 23.4380.0526 3.95 format T150 flask 150 30-45 mL 0.3125 46.875 1.64 4.11 46.880.0526 7.89 format T175 flask 175   35-52.5 0.3125 54.688 1.41 3.5254.69 0.0526 9.21 format T235 flask 235   47-70.5 0.3125 73.438 1.052.62 73.44 0.0526 12.36 format T300 flask 300 30-40 mL? 0.3125 93.7500.82 2.05 93.75 0.0526 15.78 format CF1 6/36 150-200 0.3125 198.750 0.390.0526 33.45 CF2 1272 300-400 0.3125 397.500 0.19 0.0526 66.91 CF1063360 1500-2000 0.3125 19800.000 0.00 0.0526 3332.74It is apparent that during the scale up the volume of sample per cm² ofvessel remains constant for part 1 and the same viral infectionconditions are kept in part 2.

COI assay control: The titer and back titration controls for this assaywere performed using Vero indicator cells and scored in a TCID50 96-wellformat with wells scored positive based on the media color change frompink to yellow, as a surrogate for cell death, or the presence of CPE.

Virus titer control test: Two independent replicates of the controlvirus (PRVABC59) of known titer were subjected to a 10-fold dilutionseries in media containing 2% PBS, and 100 μL of each dilution was addedto four wells of a 96-well plate containing Vero cells. Plates wereincubated for 5 days, then wells containing CPE were recorded and virustiter was calculated using the Reed-Meunch calculator.

Virus back titration control test: The control virus of known titer wasserially diluted to 200 TCID50. Two independent replicates of the 200TCID50 control virus were subjected to a 2-fold dilution series in mediacontaining 2% FBS, and 100 μL of each dilution was added to four wellsof a 96-well plate containing Vero cells. Cells were incubated for 5days, then wells containing CPE were recorded and virus titer wascalculated using the Reed-Meunch calculator.

Detailed COI Protocol:

-   -   1. First part of the assay: Vero (1.4E⁺⁰⁵ cells/mL) and Aedes        aegypti mosquito C6/36 (4E⁺⁰⁵ cells/mL) cells were seeded in        96-well plates two days prior to addition of the samples. The        Vero cells were cultured in DMEM+10% final FBS+2% L-glutamine+1%        penicillin/streptomycin at 37° C. C6/36 cells were cultured in        DMEM+10% FBS+2% L-glutamine+1% Penicillin/streptomycin+1%        nonessential amino acids at 28° C.    -   2. Three independent replicates of the 200 TCID50 control virus        (prepared in the virus back titration control test) or the DS        samples were diluted (5-fold and 10-fold dilutions) into media        containing 2% FBS.    -   3. The cells in 96-well plates were inoculated with the samples.        Prior to the infection of the cell monolayers in the 96-well        plates, the sample was vortexed to disrupt any possible        aggregation. 100 μL of each dilution was applied to each of 5        wells into two separate 96-well plates containing Vero and C6/36        cells, respectively.    -   4. Media alone was included in another well for each cell type        as a negative CPE control.    -   5. Plates were incubated for 6 days at the appropriate        temperature for the cell line.    -   6. Second part of the assay: To allow live virus to be further        amplified and visualized by CPE on a permissive cell line, the        entire volume of each 96-well supernatant from both Vero and        C6/36 cells was transferred to individual wells of 6-well plates        of Vero cells. Inoculation proceeded for 90 minutes with rocking        at 15 minutes intervals.    -   7. Medium containing 2% FBS was added to the wells and plates        were incubated for an additional 8 days for subsequent detection        of the amplified samples as a function of CPE. The inactivation        was considered to be incomplete if any of the replicates of the        DS showed CPE at the end of day 8.    -   7. The presence of live/replicating virions was visualized by        the formation of plaques or CPE on susceptible cell monolayers        after transfer to the 6-well plate, and incubation for 8 days to        allow for viral replication. The % CPE scoring in the 6-well        plates at the end of the assay was calculated as follows:        -   Each 6-well plate of Vero cells was examined for CPE by            visualization of colorimetric change, followed by            confirmation of CPE by inspection under an inverted light            microscope.        -   Each 6-well plate represented one of the replicates of the            DS dilutions prepared in the 5 and 10-fold dilutions            described above (5 wells, plus one well containing media            alone).    -    Therefore, % CPE for each replicate reflected the number of        wells with CPE out of 5 total wells per sample (120 total wells        are used per assay). Mean % CPE and standard deviation were        calculated based on three replicates of each dilution.

Results: The daily samples were analyzed in each of the Tox lots #1-4 asshown in the following tables.

TABLE A Kinetics of Inactivation, Tox lot #1 Mean Sample Transfer % CPESTDV 1:10 Day −1 Vero-to-Vero 100 0 1:10 Day 0 Vero-to-Vero 100 0 1:10Day 1 Vero-to-Vero 0 0 1:10 Day 2 Vero-to-Vero 0 0 1:10 Day 3Vero-to-Vero 0 0 1:10 Day 4 Vero-to-Vero 0 0 1:10 Day 7 Vero-to-Vero 0 01:10 Day 8 Vero-to-Vero 0 0 1:10 Day 9 Vero-to-Vero 0 0 1:10 Day 10Vero-to-Vero 0 0 100TCID50/mL Vero-to-Vero 100 0 1:10 Day −1C6/36-to-Vero 100 0 1:10 Day 0 C6/36-to-Vero 100 0 1:10 Day 1C6/36-to-Vero 6.7 12 1:10 Day 2 C6/36-to-Vero 13.3 12 1:10 Day 3C6/36-to-Vero 0 0 1:10 Day 4 C6/36-to-Vero 0 0 1:10 Day 7 C6/36-to-Vero0 0 1:10 Day 8 C6/36-to-Vero 0 0 1:10 Day 9 C6/36-to-Vero 0 0 1:10 Day10 C6/36-to-Vero 0 0 100TCID50/mL C6/36-to-Vero 100 0

TABLE B Kinetics of Inactivation, Tox lot #2 Mean Sample Transfer % CPESTDV 1:10 Day −1 Vero-to-Vero 100 0 1:10 Day 0 Vero-to-Vero 100 0 1:10Day 1 Vero-to-Vero 100 0 1:10 Day 2 Vero-to-Vero 0 0 1:10 Day 3Vero-to-Vero 0 0 1:10 Day 4 Vero-to-Vero 0 0 1:10 Day 7 Vero-to-Vero 0 01:10 Day 8 Vero-to-Vero 0 0 1:10 Day 9 Vero-to-Vero 0 0 1:10 Day 10Vero-to-Vero 0 0 100TCID50/mL Vero-to-Vero 100 0 1:10 Day −1C6/36-to-Vero 100 0 1:10 Day 0 C6/36-to-Vero 100 0 1:10 Day 1C6/36-to-Vero 100 12 1:10 Day 2 C6/36-to-Vero 13.3 12 1:10 Day 3C6/36-to-Vero 0 0 1:10 Day 4 C6/36-to-Vero 0 0 1:10 Day 7 C6/36-to-Vero0 0 1:10 Day 8 C6/36-to-Vero 0 0 1:10 Day 9 C6/36-to-Vero 0 0 1:10 Day10 C6/36-to-Vero 0 0 100TCID50/mL C6/36-to-Vero 100 0

TABLE C Kinetics of Inactivation, Tox lot #3 Mean Sample Transfer % CPESTDV 1:10 Day −1 Vero-to-Vero 100 0 1:10 Day 0 Vero-to-Vero 100 0 1:10Day 1 Vero-to-Vero 27 12 1:10 Day 2 Vero-to-Vero 0 0 1:10 Day 3Vero-to-Vero 0 0 1:10 Day 4 Vero-to-Vero 0 0 1:10 Day 7 Vero-to-Vero 0 01:10 Day 8 Vero-to-Vero 0 0 1:10 Day 9 Vero-to-Vero 0 0 1:10 Day 10Vero-to-Vero 0 0 100TCID50/mL Vero-to-Vero 100 0 1:10 Day −1C6/36-to-Vero 100 0 1:10 Day 0 C6/36-to-Vero 100 0 1:10 Day 1C6/36-to-Vero 87 12 1:10 Day 2 C6/36-to-Vero 27 12 1:10 Day 3C6/36-to-Vero 0 0 1:10 Day 4 C6/36-to-Vero 0 0 1:10 Day 7 C6/36-to-Vero0 0 1:10 Day 8 C6/36-to-Vero 0 0 1:10 Day 9 C6/36-to-Vero 0 0 1:10 Day10 C6/36-to-Vero 0 0 100TCID50/mL C6/36-to-Vero 100 0

TABLE D Kinetics of Inactivation, Tox lot #4 Mean Sample Transfer % CPESTDV 1:10 Day −1 Vero-to-Vero 100 0 1:10 Day 0 Vero-to-Vero 93 12 1:10Day 1 Vero-to-Vero 0 1:10 Day 2 Vero-to-Vero 0 0 1:10 Day 3 Vero-to-Vero0 0 1:10 Day 4 Vero-to-Vero 0 0 1:10 Day 7 Vero-to-Vero 0 0 1:10 Day 8Vero-to-Vero 0 0 1:10 Day 9 Vero-to-Vero 0 0 1:10 Day 10 Vero-to-Vero 00 100TCID50/mL Vero-to-Vero 100 0 1:10 Day −1 C6/36-to-Vero 100 0 1:10Day 0 C6/36-to-Vero 100 0 1:10 Day 1 C6/36-to-Vero 33 23 1:10 Day 2C6/36-to-Vero 7 12 1:10 Day 3 C6/36-to-Vero 0 0 1:10 Day 4 C6/36-to-Vero0 0 1:10 Day 7 C6/36-to-Vero 0 0 1:10 Day 8 C6/36-to-Vero 0 0 1:10 Day 9C6/36-to-Vero 0 0 1:10 Day 10 C6/36-to-Vero 0 0 100TCID50/mLC6/36-to-Vero 100 0

Compiled kinetics of inactivation data: COI data for samples from thefour toxicology lots were compared to infectious potency (TCID50)determined as described above and to RNA copy. The RNA copy wasdetermined by purifying nucleic acids from the sample and amplifyingZika RNA with serotype-specific primers using an RT-PCR kit. The resultshown in FIG. 25 shows that the sensitivity of the COI assay issignificantly greater than that of TCID50.

Performance characteristics of the COI assay—Accuracy: The targetdilutions (TCID50/well) and their respective proportions of CPE wereused to determine relative accuracy. For the Vero cells, there was astatistically significant linear relationship between the observed andexpected proportions of positive CPE. The slope of the line relatingobserved and expected results is 0.92 with a 95% confidence interval(CI) of 0.83 to 1.01 that overlaps 1 indicate 100% accuracy. For theC6/36 cells, there is a statistically significant linear relationshipbetween the observed and expected proportions of positive CPE. The slopeof the line relating observed and expected results is 0.88 with a 95%confidence interval (CI) of 0.80 to 0.95 indicate that a slight bias(5-20%) was seen with this cell line. Both cell lines demonstratesatisfactory accuracy (relative).

Performance characteristics of the COI assay—Limit of Detection (LoD):The sensitivity of the assay was assessed for both the C6/36-to-Vero andVero-to-Vero plates. As described above, the data was fitted using leastsquares regression of the proportion of +ve CPE observed per total wellsplated with titer dilutions plated starting at 10.00 TCID50/well down toa lower titer of 0.08 TCID50. Furthermore, negative controls (0.00TCID50/well) were included for each dilution within the plates. CPEscoring was performed for each dilution across both the C6/36-to-Veroand Vero-to-Vero plates. A clear relationship between the CPE and loginput virus titer was seen. This displays the logistic (sigmoidal)relationship between the proportion of CPE positive wells relative tothe log₁₀ concentration of TCID50/well together with a lower and upper99% confidence limit. At a −2 log₁₀ concentration (=0.01 TCID50/well), amodel based on and accounting for all fixed and random sources variationin the qualification data predicted 0.85%, or 0.01 when rounded up at0.01 TCID50/well, with a lower 99% confidence limit of 0.42%. Since thelower 99% confidence limit does not include zero, there is a very smallquantifiable (<1%) chance the 0.85% CPE wells could have arisen from 0TCID50/well (i.e., due to noise). This establishes a detection limit forthe assay of at least 0.01 TCID50/well (i.e., the lowest amount of liveZika particles in the sample which can be detected). That is, whenrounded up, 1 in 60 wells will be CPE positive or given theseparameters, the lowest theoretical proportion of the CPE+ve that couldbe detected in 60 wells would be 1.67%, or 0.0167.

The cell types (C363 and Vero) were compared for relative sensitivity,with the C6/36 demonstrating that a lower dilution of virus titer couldbe detected compared to Vero cells as shown in FIG. 26 ; at the samevirus input level (0.31 TCID50), the proportion of CPE positive wells ishigher for C6/36 relative to Vero cells.

The lowest virus input value used during the qualification of this assaywas 0.02 TCID50 (−1.61 log TCID50). Using the fitted curve for C6/36cells, this results in 0.035 or 3.5% of the wells scoring CPE positive(1 in 28 wells). If the curve is extrapolated towards the lowestpractical level of 0.167 or 1.6%, then this equates to a virus inputlevel of 0.015 TCID50 (−1.82 log TCID50). However, the impact oftransmitted assay variance needs to be considered when determining thelowest levels of infectious virus that can be detected as reflected inthe +ve CPE results. This noise arises from generation of the workingstock of input virus. Comparison of the target TCID50 and theback-titration calculation shows the TCID50 of the working stock virusexhibited a standard deviation (SD) of 85 TCID50/mL, derived from a meanof 213 when targeting a stock TCID50/mL concentration of 200. The % CVcalculates to ˜40% with a bias of about +7%. This noise was factoredinto the logistic regression model to generate confidence intervalsaround the targeted values for the virus dilutions. At a target value of0.01 TCID50/well, a model based on and accounting for all fixed andrandom sources of variation in the qualification data across the twosites predicts 0.86% of wells will be CPE positive (1 in 60 wells).Since the lower 99% confidence limit does not include zero, there is avery small quantifiable (<1%) chance the 0.85% CPE-positive wells couldhave arisen from 0 TCID50/well due to noise (FIG. 27 ). This establishesa detection limit for the assay: 0.01 TCID50/well is the lowest amountof live Zika particles in the sample which can be detected.

Performance characteristics of the COI assay—Range: The range of theassay was 0.01 TCID50/well to 4.5 TCID50/well and is defined as therange of input virus that resulted in a CPE+ve proportion scoring ofmore than 0% but less than 100%.

Conclusion: Analysis of the four Tox revealed that inactivation wascomplete after incubation in 0.01% formaldehyde for 10 days at roomtemperature. Inactivation was achieved by days 3-4 in all lots produced,as measured by the COI assay. The COI assay is more sensitive thanTCID50 potency or RNA measurements; the increased sensitivity has alsobeen observed by LoD.

Example 5: Determining Residual Formalin Content in a PharmaceuticalComposition Materials and Methods Materials

Formaldehyde standard solution (in methanol) (982 μg/mL), DNPH,HPLC-grade acetonitrile, and phosphoric acid were purchased from WakoPure Chemicals Co. (Tokyo, Japan). Distilled water used for dilutingphosphoric acid was obtained from Otsuka Pharmaceutical (Tokushima,Japan). Alhydrogel® 2% (corresponding to 10 mg/mL aluminum) used asaluminum hydroxide gel was obtained from Brenntag (Frederikssund,Denmark). PBS was prepared in-house, and the Zika vaccine drug productcontaining aluminum hydroxide gel was manufactured as described below.The Zika virus was purified with various techniques after harvest. Afterinactivation with formaldehyde, the virus was concentrated, and thebuffer was exchanged with PBS by filtration. The bulk drug substance wasdiluted with PBS and formulated with aluminum hydroxide gel (0.4 mg/mLaluminum) to form the final drug product.

HPLC Conditions

A Waters HPLC alliance system equipped with a UV detector (Milford, USA)and a reverse-phase column (YMC-Pack ODS-A, 4.6 mm×250 mm, 5 μm (Kyoto,Japan)) was used. A mixture of water and acetonitrile (1:1, v/v) wasused as the mobile phase, the detection wavelength was set at 360 nm,and the flow rate was 1.0 mL/min. The column temperature and injectionvolume were 25° C. and 50 μL, respectively.

Sample Preparation

The vaccine drug product (1.2 mL) was centrifuged at 15000 rpm for 10min, and the supernatant (1 mL) was transferred into a 2-mL HPLC glassvial purchased from Waters (Milford, USA). Next, 20 μL of 20% (v/v)phosphoric acid and 50 μL of 1.0 mg/mL DNPH solution in acetonitrilewere added, and the mixture was stirred and left at room temperature for20 min before injection.

Method Validation

According to the ICH Q2 guidelines, the method was validated in terms ofspecificity, linearity, accuracy, repeatability, intermediate precision,robustness, and stability of the sample. In the accuracy study, the Zikavaccine drug product and aluminum hydroxide gel solution were spikedwith a specific amount of formaldehyde, and the sample was mixed well byvortex before following the procedure described in Section 2.3.

Results and Discussion Linearity and Specificity

Six standard solutions of formaldehyde (0.049, 0.098, 0.196, 0.491,0.982, and 1.964 μg/mL) were prepared by dilution with PBS. Next, 20%(v/v) phosphoric acid and 1 mg/mL DNPH solution in acetonitrile wereadded to each solution, and the corresponding chromatograms are shown inFIG. 28 . Clearly, the 10.4-min peak area showed linearity with theregression equation: y=1075730x+11731 (where y is the area of the10.4-min peak and x is the concentration of formaldehyde in μg/mL)(correlation coefficient: 0.9998), indicating that it was due toHCHO-DNPH (i.e., formaldehyde derivatized with DNPH). Moreover, the peakat 5.8 min was attributed to DNPH as it was detected in all samplesadded with DNPH. Hence, the HCHO-DNPH peak area was used for evaluationof linearity and accuracy after subtracting the background peak area inPBS.

Accuracy and Precision (Repeatability)

The effect of aluminum hydroxide adjuvant was evaluated by recoverystudies, which were carried out by spiking three samples of aluminumhydroxide (0.1, 0.4, and 1.0 mg/mL aluminum) in PBS with 0.05 μg/mL offormaldehyde in the absence of the vaccine drug substance. The averagerecoveries were 102% (n=3), 100% (n=3), and 100% (n=3), respectively,with low relative standard deviation (RSD) values (Table 13). The RSD ofthe accuracy data was calculated to evaluate the repeatability, and wasfound to be 1.0%, indicating that aluminum amounts up to 1.0 mg/mL didnot interfere with the recovery of formaldehyde.

TABLE 13 Accuracy and repeatability evaluated using aluminum hydroxidesamples spiked with 0.05 μg/mL of formaldehyde Aluminum hydroxideAverage (n = 3) concentration [%] [mg/mL aluminum] (RSD [%]) 0.1 102(0.2) 0.4 100 (0.8) 1.0 100 (0.3) Repeatability [%] (n = 9) 1.0

The accuracy of the method was evaluated by recovery studies, which werecarried out by spiking the Zika vaccine drug product containing aluminumhydroxide adjuvant with three concentrations of formaldehyde (0.05,0.10, and 1.00 μg/mL), and the average recovery results are shown inTable 14. The RSD of the accuracy data was calculated to evaluate therepeatability, and was found to be 3.7%, indicating that Zika vaccinedrug products formulated with aluminum hydroxide do not interfere withthe recovery of formaldehyde between 0.05 and 1.00 μg/mL.

TABLE 14 Accuracy and repeatability evaluated using Zika vaccine drugproducts containing aluminum hydroxide spiked with formaldehyde Spikedformaldehyde Average (n = 3) concentration [%] [mg/mL] (RSD [%]) 0.05102 (5.6) 0.10 97 (0.3) 1.00 98 (0.7) Repeatability [%] (n = 9) 3.7

Robustness

The robustness of the method was evaluated to determine howconcentration of formaldehyde in samples would be affected by variationsin experimental parameters during sample preparation. Considering impacton the derivatization efficacy, concentration of DNPH and phosphoricacid were selected as the monitored parameters in this study. The effectwas examined by varying the concentrations of DNPH and phosphoric acidby ±0.1 mg/mL and ±5%, respectively. Formaldehyde was determined in twodevelopment drug product lots under each condition, and the results,shown in Table 15, suggest that variations in DNPH and phosphoric acidconcentrations had no significant impact on the determination offormaldehyde.

TABLE 15 Robustness of the method Concentration Concentration ofConcentration of of DNPH phosphoric acid formaldehyde [μg/mL] Condition[mg/mL] [%] Lot B Lot C 1* 1.0 20 0.51 0.45 2 1.1 20 0.53 0.48 3 0.9 200.49 0.47 4 1.0 15 0.52 0.49 5 1.0 25 0.52 0.48 *Defined conditions ofthe method

Example 6: Clinical Immunogenicity and Efficacy of a PurifiedInactivated Zika Virus Vaccine (PIZV) Derived from P6e Strains SamplePreparation

Four Purified Inactivated Zika Vaccine (PIZV) lots (Tox lots 1-4) weremanufactured by growth in Vero cells as described above. Supernatantsfrom 4 daily harvests (each daily harvest 1000 mL daily, totaling about4000 mL) were purified by filtration and chromatography, concentratedand inactivated by addition of formalin to a final concentration of0.01%. Inactivation was allowed to proceed for 10 days at 22° C., beforethe sample was buffer exchanged into Drug Substance Buffer (10 mMNaH2PO4, 50 mM NaCl, 6% sucrose, pH 7.4).

The inactivated Zika virus active agent is no longer able to infect hostcells, which can be infected with a Zika virus, which has not beeninactivated. The inactivation is determined by the following testprotocol. Inactivation is acknowledged in case no plaques aredetectable.

Detailed COI (Completeness of Inactivation) protocol:

-   -   1. First part of the assay: Vero (1.4E⁺⁰⁵ cells/mL) and Aedes        aegypti mosquito C6/36 (4E⁺⁰⁵ cells/mL) cells were seeded in        96-well plates two days prior to addition of the samples. The        Vero cells were cultured in DMEM+10% final FBS+2% L-glutamine+1%        penicillin/streptomycin at 37° C. C636 cells were cultured in        DMEM+10% FBS+2% L-glutamine+1% Penicillin/streptomycin+1%        nonessential amino acids at 28° C.    -   2. Three independent replicates of the 200 TCID50 control virus        (prepared in the virus back titration control test) or the DS        samples were diluted (5-fold and 10-fold dilutions) into media        containing 2% FBS.    -   3. The cells in 96-well plates were inoculated with the samples.        Prior to the infection of the cell monolayers in the 96-well        plates, the sample was vortexed to disrupt any possible        aggregation. 100 μL of each dilution was applied to each of 5        wells into two separate 96-well plates containing Vero and C636        cells, respectively.    -   4. Media alone was included in another well for each cell type        as a negative CPE control.    -   5. Plates were incubated for 6 days at the appropriate        temperature for the cell line.    -   6. Second part of the assay: To allow live virus to be further        amplified and visualized by CPE on a permissive cell line, the        entire volume of each 96-well supernatant from both Vero and        C636 cells was transferred to individual wells of 6-well plates        of Vero cells. Inoculation proceeded for 90 minutes with rocking        at 15 minutes intervals.    -   7. Medium containing 2% FBS was added to the wells and plates        were incubated for an additional 8 days for subsequent detection        of the amplified samples as a function of CPE. The inactivation        was considered to be incomplete if any of the replicates of the        DS showed CPE at the end of day 8.    -   7. The presence of live/replicating virions was visualized by        the formation of plaques or CPE on susceptible cell monolayers        after transfer to the 6-well plate, and incubation for 8 days to        allow for viral replication. The % CPE scoring in the 6-well        plates at the end of the assay was calculated as follows:        -   Each 6-well plate of Vero cells was examined for CPE by            visualization of colorimetric change, followed by            confirmation of CPE by inspection under an inverted light            microscope.        -   Each 6-well plate represented one of the replicates of the            DS dilutions prepared in the 5 and 10-fold dilutions            described above (5 wells, plus one well containing media            alone).    -    Therefore,% CPE for each replicate reflected the number of        wells with CPE out of 5 total wells per sample (120 total wells        are used per assay). Mean % CPE and standard deviation were        calculated based on three replicates of each dilution.

The amount of the purified inactivated Zika virus can be determined by aBradford assay (Bradford et al. (1976) Anal. Biochem. 72: 248-254) usingdefined amounts of recombinant Zika envelope protein to establish thestandard curve.

The purity of the purified Zika virus can be determined by sizeexclusion chromatography. In the current example, the main peak of thepurified Zika virus in the size exclusion chromatography was more than85% of the total area under the curve in the size exclusionchromatography.

The investigational vaccine (PIZV) refers to Zika purifiedformalin-inactivated virus formulated with 200 μg aluminum hydroxide,Al(OH)3, as adjuvant, in phosphate buffered saline solution (PBS). Thefinal liquid formulated product is filled into single-use vials andsealed with tamper-evident seals. The investigational vaccine isadministered IM (intramuscularly) as a 2-dose regimen of 0.5 mL at 2, 5,or 10 μg antigen per dose, 28 days apart.

Sodium chloride (NaCl) 0.9% solution for injection is being used asplacebo. It is supplied in single-use vials. It is a sterile, clear,colorless liquid solution of sodium chloride without preservativedesigned for parenteral use only. The placebo is administered IM as a2-dose regimen of 0.5 mL per dose, 28 days apart.

Test Methods

PRNT assay: Neutralizing antibody titers were determined by a plaquereduction neutralization test (PRNT) as described previously (SeeProtection of Rhesus monkeys against dengue virus challenge aftertetravalent live attenuated dengue virus vaccination. J. Infect. Dis.193, 1658-1665 (2006). Muthumani K, Griffin B D, Agarwal S, et al. Invivo protection against ZIKV infection and pathogenesis through passiveantibody transfer and active immunisation with a prMEnv DNA vaccine. NPJVaccines 2016; 1: 16021). Briefly the Zika PRNT assay was carried outaccording to the protocol developed by Q2 Lab Solutions Vaccines asdescribed below.

In the Zika PRNT assay, human serum was 2-fold serially diluted from 1:5to 1:10,240 and mixed with an equal volume of diluted Zika virus (ZIKV)(PRVABC59) to obtain a final dilution of 1:10 to 1:20,480.Neutralization was allowed to proceed 20±2 hours at 2-8° C. after whichthe serum/virus mixture was used to inoculate Vero E6 cells. Virusadsorption was done at 37±2° C. with humidity and CO₂ for 60±10 minutesthen a methylcellulose overlay was added. The infected cells wereincubated at 37±2° C. with humidity and CO₂ for 72±2 hours. Plaques werevisualized by using crystal violet staining and were counted using a CTL(Cellular Technology Limited) reader. Determination of the fifty percentneutralizing titer (PRNT₅₀) was based upon the percent reduction inviral plaques in the presence of serum compared to that of the viruscontrol without serum and was calculated by linear regression. Thetiters represent the reciprocal of the highest dilution resulting in a50% reduction in the number of plaques. Acceptance was assessed byevaluating the virus control (targeting ˜60 pfu/well), cell control,positive control (PRNT₅₀ of 173-658) and negative control (PRNT₅₀<10)tested in parallel with clinical samples. Individual samples andpositive control results were accepted if the correlation coefficient ofthe titration curve generated by linear regression is ≥0.85. Additionalacceptance criteria were based on the quality of the crystal violetstain and plaques generated for the plate or run. PRNT₅₀ results arereported down to the starting dilution of the assay (1:10). PRNT₅₀results that are above the ULOQ will be repeated at a pre-dilution togenerate a result within the quantifiable range of the assay. The resultfrom the pre-diluted sample will be multiplied by the dilution factor togenerate a final result.

The Zika strain used for PRNT assay development was PRVABC59.

Immuno Outcome Measures Definitions

Sero-positivity (PRNT): titers of ≥LOD (Limit of detection)

Sero-negativity (PRNT): titers of <LOD (Limit of detection)

Seroconversion (PRNT): Post vaccination titers of ≥LOD in initiallyseronegative human subjects

LOD (PRNT)=10

Assigned value for below LOD=5

LLoQ (Lower Limit of Qualification, PRNT)=26

Assigned value for below LLoQ (Lower Limit of Qualification)=13

Human subjects with titers above the Upper Limit of Quantitation (ULOQ)will be retested after further dilution until a titer is obtained withinthe assay qualified limit of quantitation.

Reporter virus particle (RVP) neutralization assay: Neutralizingantibody titers were analyzed by titration of serum samples with aconstant amount of Zika RVPs in Vero cells grown in 96-well plates. RVPscontained the prME proteins of Zika (strain SPH2012) and a Dengue-basedRenilla luciferase reporter. Briefly, sera were heat inactivated at 56°C. for 30 min, diluted, and then incubated at 37° C. with RVPs. Theserum/RVP mixture was then mixed with Vero cells and incubated for 72hours at 37° C.±2° C./5% CO2 before detection with luciferase substrate.Data was analyzed using JMP11 non-linear 4 parameter analysis,normalized to a positive tracking control and effective dose 50% (EC50)was reported.

Study Description

A Phase 1, Randomized, Observer-Blind, Placebo-Controlled, Safety,Immunogenicity, and Dose Ranging Study of Purified Inactivated ZikaVirus Vaccine (PIZV) in Flavivirus Naïve and Primed Healthy Adults Aged18 to 49 Years

The study design is shown in FIG. 29 . This study was designed tosequentially enroll flavivirus-naïve and flavivirus-primed healthyadults between the ages of 18 and 49 years. The two sequential cohortsare each comprised of 120 human subjects (planned) randomly allocated toone of 4 groups of 30 human subjects, to receive either one of threedosages of the PIZV vaccine or saline placebo. The vaccination regimenconsists of 2 doses administered 28 days apart. The data in this exampleonly relates to the Flavivirus naïve human subjects (n=124), furtherdata with Flavivirus primed human subjects are to be expected. Thisexample provides data from a first interim analysis following Day 57 (28days post-dose 2) for the “flavivirus-naïve cohort”. Data from theflavivirus-primed cohort are not part of this interim analysis, asrecruitment for this group was still ongoing at the time first interimanalysis.

In summary, human subjects were randomized into four study groups, whoreceived two doses of either placebo (saline) or purified inactivatedZika vaccine (PIZV) with a concentration of 2 μg, 5 μg and 10 μg. Thestudy involved intramuscular injection of the vaccine (or placebo) atday 1 and day 29, with blood samples being taken on day −15, 1, 8, 29,36, 57, 211, 393, 767 of the study. Blood samples on day −15 were usedto determine Flavivirus serostatus screening and eligibility screening.Samples on day 1, 29, 57 were for immunogenicity assessment. Safety labtesting was carried out on days 8 and 36. Persistence of immunity willbe assessed on day 211, 393 and 767.

Based on the data from 28 days post dose 2, the purified inactivatedZika virus vaccine (PIZV) was safe and immunogenic in Flavivirus-naïveadults aged between 18-49 yrs.

Primary Objectives

The primary objective of the study was to describe the safety of twodoses of PIZV given 28 days apart and to select a dose level from threedifferent antigen concentrations (2, 5 or 10 μg) for use in subsequentclinical studies. The primary endpoints were: the percentages of humansubjects experiencing solicited local and systemic adverse events (AEs)during the 7-day period after administration of each dose of PIZV orplacebo, and the percentages of human subjects experiencing non-seriousunsolicited AEs and serious adverse events (SAEs) during the 28-dayperiod after vaccination.

Secondary Objectives

The secondary objectives were to describe the immune response to thepurified inactivated Zika virus vaccine (PIZV) at 28 days post dose 1and 28 days post dose 2 in flavivirus naïve adults. The secondaryendpoints related to these objectives are geometric mean titers (GMTs)of neutralizing anti-ZIKV antibodies, seropositivity rates (SPR) andseroconversion rates (SCR) at the considered timepoints.

Analysis of the data was performed by a separate set of unblindedstatisticians and programmers, who had access to the individualtreatment assignments. All personnel involved in the conduct of thetrial were blinded to the individual human subject treatmentassignments. The study team had access to the group level unblindedresults only.

Study Population

A total of 124 human subjects were enrolled in the flavivirus-naïvecohort and included in the Safety Set (SS), comprised of all randomizedhuman subjects who have received at least one dose of PIZV or placebo.Among those, 118 (95.2% of the SS) were included in the Full AnalysisSet (FAS) of randomized human subjects who had received at least onedose of the investigational vaccine (PIZV)/placebo, provided validserology results at baseline and at least once post-vaccination. Onehundred and thirteen (113) human subjects (91.1% of the SS) wereincluded in the Per Protocol Set (PPS) of human subjects in the FAS whohad no major protocol violations relevant for the immunogenicityanalysis. The analysis sets are presented in Table 16.

TABLE 16 Analysis sets Number of Human subjects (%) Placebo 2 μg PIZV 5μg PIZV 10 μg PIZV Total (N = 30) (N = 31) (N = 31) N = 32) (N = 124)Safety Set (SS) 30 (100%) 31 (100%) 31 (100%) 32 (100%) 124 (100%) FullAnalysis 29 (96.7%) 28 (90.3%) 31 (100%) 30 (93.8%) 118 (95.2%) Set(FAS) Per-Protocol 28 (93.3%) 26 (83.9%) 29 (93.5%) 30 (93.8%) 113(91.1%) Set (PPS) Safety Set = all randomized human subjects whoreceived at least one (1) dose of PIZV or placebo Full Analysis Set =all randomized human subjects who received at least one dose ofPIZV/placebo and provided valid baseline and at least onepost-vaccination serology result Per Protocol Set = all human subjectsin the FAS who had no major protocol violations

Human subjects in the SS were 35.3±8.91 years of age (mean±standarddeviation), and were distributed as 28.2% in the 18-29 years age-rangeand 71.8% in the 30-49 years age-range. Women represented 54.8% of thecohort. Study participants were White (81.5%), Black (14.5%), and“Non-Hispanic” (93.5%) regarding race and ethnicity. The mean BMI in theSS was 27.5±4.05 (mean±standard deviation). Demographic characteristics(age, sex, height, weight, BMI and ethnicity) were overall similaracross the four study groups. Women were more represented in the placebogroup, where they constituted 66% of the study participants, than in theother groups, where gender distribution was more balanced. Thedemographics and baseline characteristics are presented in Table 17.

Safety laboratory parameters and vital signs were checked at study entryas part of inclusion criteria. These specified that vital signs had tobe within normal limits (i.e., below Grade 1 as indicated in the FDAToxicity Grading Scale) and that safety laboratory tests had to bewithin normal limits or not be above Grade 1 as defined in the FDAToxicity Grading Scale.

TABLE 17 Demographic and Baseline Characteristics (Safety Set) Number ofHuman subjects (%) Placebo 2 ug 5 ug 10 ug Total (N = 30) (N = 31) (N =31) (N = 32) (N = 124) Age (Years) n 30 31 31 32 124 Mean (SD) 36.5(9.00) 34.9 (9.52) 35.8 (8.86) 34.1 (8.50) 35.3 (8.91) Median 39.5 36.036.0 33.5 36.0 Minimum, 18, 49 18, 48 20, 49 20, 49 18, 49 Maximum Age(years) (n [%]) 18-29   8 (26.7)   9 (29.0)   9 (29.0)   9 (28.1)   35(28.2) 30-49   22 (73.3)   22 (71.0)   22 (71.0)   23 (71.9)   89 (71.8)Sex (n [%]) Male   10 (33.3)   15 (48.4)   13 (41.9)   18 (56.3)   56(45.2) Female   20 (66.7)   16 (51.6)   18 (58.1)   14 (43.8)   68(54.8) Ethnicity (n [%]) Hispanic   1 (3.3)   1 (3.2)   2 (6.5)   4(12.5)   8 (6.5) or Latino Not-Hispanic   29 (96.7)   30 (96.8)   29(93.5)   28 (87.5)  116 (93.5) or Latino Not Reported  0  0  0  0  0Unknown  0  0  0  0  0 Race (n [%]) American   2 (6.7)  0  0  0   2(1.6) Indian or Alaskan Native Asian  0  0  0  0  0 Black or   6 (20.0)  5 (16.1)   3 (9.7)   4 (12.5)   18 (14.5) African American Native  0 0  0  0  0 Hawaiian or Other Pacific Islander White   22 (73.3)   26(83.9)   26 (83.9)   27 (84.4)  101 (81.5) Multiracial  0  0   2 (6.5)  1 (3.1)   3 (2.4) BMI (kg/m²) n 30 31 31 32 124 Mean (SD) 28.16927.527 27.541 26.750 27.485 (4.0388) (4.7632) (3.7165) (3.6511) (4.0452)Median 28.861 27.900 27.831 25.965 27.607 Minimum, 20.86, 20.13, 18.84,18.80, 18.80, Maximum 34.64 34.83 34.14 34.37 34.83 BMI = Weight(kg)/height² (m²). Note 1: Age is calculated using the Date of InformedConsent. Note 2: Human subject included in Multiracial Category only ifmultiple Race categories selected.

Safety/Reactogenicity

The overall reporting incidence of solicited local adverse events (AEs)was higher in the groups that received the vaccine (PIZV) than in theplacebo group. Pain was the most frequently reported solicited AE at theinjection site. After dose 1, pain was experienced by 30.0% to 38.7% ofhuman subjects in the PIZV groups compared to 13.8% in the placebogroup. After dose 2, incidences of pain were similar to those followingdose 1: 29.6% to 40% in the PIZV groups, and 14.3% in the placebo group.Intensity of pain was reported as mild after dose 1 and mild to moderateafter dose 2, with 2 human subjects in the 5 μg PIZV group (6.7%) andone human subject in the 10 μg PIZV group (3.3%) reporting moderatepain. Other solicited local AEs (erythema, swelling and induration) werereported by not more than 9.7% of the human subjects.

The onset of pain occurred on day 1 for 90% of the human subjects or day2 (for 3 human subjects). Pain was not reported beyond day 5 by anyhuman subject in the placebo or PIZV groups.

Solicited systemic AEs of any nature were reported by 30% to 48.4% ofthe human subjects across the PIZV groups and by 41.4% in the Placebogroup after dose 1. After dose 2, incidences were 10% to 33.3% acrossthe PIZV groups and 27.6% in the Placebo group. Overall 81.3% (dose 1)and 75% (dose 2) of the solicited systemic AEs were judged as related tostudy vaccination. After both doses, the most reported systemic eventswere headache, fatigue and myalgia.

Most systemic AEs were reported as mild, i.e. not interfering with dailyactivity. A few occurrences were moderate in intensity:

after dose 1, for 6.7-12.9% of human subjects in the PIZV groups and17.2% of placebo recipients;

after dose 2, for 0-3.3% of human subjects across the PIZV groups and10.3% in the placebo group.

There was a single report of a severe AE: one human subject in theplacebo group experienced fever. This study participant presented atemperature of 39.4° C., measured orally, 4 days after receiving thesecond study vaccination. This fever was not judged as study-related bythe investigator.

Solicited systemic AEs were variably reported throughout the 7-dayperiod in the four groups. The onset of events for fever, fatigue,arthralgia and myalgia was mainly during the 2 days followingvaccination and was variable for headache and malaise. Fever wasreported during the 2 days following vaccination, except for the humansubject reporting severe fever in the placebo group on day 4.

The incidence of solicited local and systemic adverse events 7 daysafter vaccination are shown in Table 18.

TABLE 18 Incidence of solicited local and systemic adverse events 7 daysafter vaccination (Safety set) Dose 1 Dose 2 2 μg 5 μg 10 μg 2 μg 5 μg10 μg Placebo PIZV PIZV PIZV Placebo PIZV PIZV PIZV (N = 30) (N = 31) (N= 31) (N = 32) (N = 30) (N = 31) (N = 31) (N = 32) Local AEs n (%) Any 4(13.8) 9 (30.0) 12 (38.7) 13 (41.9)  5 (17.9) 8 (29.6) 11 (36.7) 12(40.0) Pain 4 (13.8) 9 (30.0) 10 (32.3) 12 (38.7)  4 (14.3) 8 (29.6) 11(36.7) 12 (40.0) Erythema 0 0 0 1 (3.2) 1 (3.6) 0 1 (3.3) 1 (3.3)Swelling 0 0 0 0 0 0 2 (6.7) 0 Induration 0 0 3 (9.7) 2 (6.5) 0 0 1(3.3) 0 Systemic AEs n (%) Any 12 (41.4)  9 (30.0) 12 (38.7) 15 (48.4) 8 (27.6) 9 (33.3)  3 (10.0)  8 (26.7) Fever 1 (3.4)  0 0 1 (3.2) 2(7.1) 0 0 0 Headache 9 (31.0) 5 (16.7)  8 (25.8)  4 (12.9)  3 (10.3) 4(14.8) 1 (3.3)  6 (20.0) Fatigue 6 (20.7) 7 (23.3)  6 (19.4) 10 (32.3) 6 (20.7) 3 (11.1) 2 (6.7)  5 (16.7) Arthralgia 1 (3.4)  1 (3.3)  1(3.2) 3 (9.7) 1 (3.4) 2 (7.4)  0 1 (3.3) Myalgia 3 (10.3) 3 (10.0)  5(16.1)  4 (12.9) 2 (6.9) 2 (7.4)  1 (3.3) 2 (6.7) Malaise 4 (13.8) 2(6.7)  2 (6.5)  4 (12.9) 2 (6.9) 0 0  3 (10.0) N = number of humansubjects with information available; n (%) = number (percentage) ofhuman subjects reporting a specific AE.

In total 30.6% of the human subjects reported unsolicited AEs (notincluding prolonged solicited AEs) in the 28 days following any dose:21.9-38.7% in the PIZV groups and 36.7% in the placebo group. These AEswere mainly infections, infestations (13.7%) and nervous systemdisorders (3.2%: headache, migraine, dizziness). All were mild tomoderate in intensity.

Unsolicited AEs were considered as related to the study vaccination forthree human subjects (2.4%). The events reported were:

at post dose 1, dizziness for one human subject in the 5 μg PIZV groupand flushing for one human subject in the 2 μg PIZV group;

at post dose 2, eye pruritus and lacrimation increased for one humansubject in the 10 μg PIZV group.

These were mild to moderate in intensity, started on day 1 or 2 aftervaccination, had a duration of 1 to 3 days and were all resolved.

One human subject discontinued with the study vaccination due to aheadache after dose 1. This human subject received PIZV and experiencedthe headache 1 day after vaccination. The headache was resolved 36 daysafter its onset. No serious adverse event (SAE) was reported during theperiod from dose 1 up to 28 days post dose 2.

The few changes from the baseline observed for blood safety laboratoryparameters in the 7 days following vaccination, e.g. from normal to mildor from mild to moderate AEs, occurred in comparable percentages ofhuman subjects across the four groups. Urinalysis parameters were eithernormal at all time-points or the grading category was similar acrossgroups and visits.

Immunogenicity

Table 19 presents the geometric antibody titers of Zika virusneutralizing antibodies (EC50) as measured by PRNT as well asseropositivity rates and seroconversion rates after each vaccine dose.

The PIZV vaccine was immunogenic in flavivirus-naive adults. All humansubjects were seronegative at baseline. Vaccination of human subjectsinitially seronegative for antibodies against Zika virus elicitedseropositivity in all human subjects after two doses of PIZV vaccine ofany dosage: seroconversion rates ranged from 69.23% to 96.43% post-dose1 and were 100% post-dose 2. All human subjects in the placebo groupremained seronegative throughout the period considered.

A dose-ranging effect was observed on seropositivity rates post-dose 1and on GMTs after each dose. After the first dose, almost all humansubjects (96.4%) who had received the 10 μg PIZV dose had mountedneutralizing antibodies against the Zika virus. The second dose led to amore than 10-fold increase in GMT from dose 1, in the three PIZV groups.

TABLE 19 Seropositivity, seroconversion rates and GMTs (Geometric meantiters) of Zika virus neutralizing antibodies (EC₅₀) (PRNT) before and28 days after administration of each dose of PIZV (Per Protocol Set)Placebo 2 μg PIZV 5 μg PIZV 10 μg PIZV (N = 28) (N = 26) (N = 29) (N =30) Sero- Pre-dose 0 0 0 0 positivity 1 rate Post-dose 0 69.23 (48.21,82.14 (63.11, 96.43 (81.65, (95% CI) 1 85.67) 93.94) 99.91) Post-dose 0100 (85.75, 100 (87.66, 100 (87.66, 2 100.00) 100.00) 100.00) Sero-Post-dose 0 69.23 (48.21, 82.14 (63.11, 96.43 (81.65, conversion 185.67) 93.94) 99.91) rate Post-dose 0 100 (85.75, 100 (87.66, 100(87.66, (95% CI) 2 100.00) 100.00) 100.00) GMTs Pre-dose 5.00 5.00 5.005.00 (95% CI) 1 Post-dose 5.00 38.06 (17.53, 93.76 (44.34, 291.41(161.74, 1 82.66) 198.30) 525.06) Post-dose 5.00 1100.75 (741.07,1992.33 (1401.28, 3689.89 (2676.75, 2 1635.00) 2832.70) 5086.49) N =number of human subjects in the PPS with PRNT data available;Seropositivity is defined as titer ≥10; Seroconversion is defined as:seronegative human subjects at baseline (titer <10) have titer ≥10post-vaccination; Results <10 are assigned a titer of 5; Titers ≥10(limit of detection) and <26 (lower limit of quantification) areassigned a value of 13.

The Geometric mean titers determined using PRNT, according to table 19,are shown graphically in FIG. 30 . The percentage of human subjectsachieving seroconversion determine using PRNT according to table 19, areshown graphically in FIG. 31 .

The distribution of neutralization titers, after dose 1 and after dose2, are shown in reverse cumulative distribution curves in FIGS. 32 and33 respectively.

In addition to measuring immune response with the PRNT assay, thesamples were also tested with the RVP neutralization assay. Table 20presents the geometric antibody titers of Zika virus neutralizingantibodies (EC50) as measured by the RVP assay. The RVP assay resultsshow a similar dose-ranging effect of the PRNT data, with graduallyhigher GMTs with increasing PIZV doses.

TABLE 20 GMTs (Geometric mean titers) of Zika virus neutralizingantibodies (EC₅₀) (RVP) before and 28 days after vaccination (PerProtocol Set) Placebo 2 μg PIZV 5 μg PIZV 10 μg PIZV Group (N = 27) (N =26) (N = 28) (N = 30) GMTs Pre-dose 1 34 (27, 43)  34 (26, 44)  32 (27,39)   46 (39, 53) (95% CI) Post-dose 1 28 (22, 36)  360 (242, 536)  656(442, 972)  1310 (875, 1961) Post-dose 2 31 (25, 40) 3148 (1988, 4986)6212 (4126, 9354) 13604 (9560, 19359) N = number of human subjects inthe PPS with RVP data available.

Conclusion

The PIZV vaccine was well tolerated and safe for all antigen dosesevaluated in the flavivirus-naive cohort. Solicited systemic AEs werereported in all groups with no apparent increase with increasing dosestrength and intensity was mild to moderate. Local solicited AEsreported were also mild to moderate in intensity across the groups.Unsolicited symptoms were reported with similar frequencies in the fourstudy groups. Overall, the vaccine was immunogenic in flavivirus-naivehuman subjects and a positive dose-ranging response was observed.

Further Items of the Invention:

-   -   1. Vaccine or immunogenic composition for use in a method of        treating or preventing, in particular preventing Zika virus        infection in a human subject population in need thereof,        comprising administering to individual human subjects of said        human subject population the vaccine or immunogenic composition        comprising antigen from a Zika virus, wherein the vaccine or        immunogenic composition is administered as a single dose or        prime administration, and wherein the administration of the        vaccine or immunogenic composition induces 14 and/or 28 days        after the single dose or prime administration geometric mean        neutralizing antibody titers in a population of at least 20        flavivirus naïve human subjects and/or in a population of at        least 20 Zika virus seronegative human subjects of greater than        10, or greater than 50, or greater than 100, or greater than        200, or greater than 250, as determined by the plaque reduction        neutralization test (PRNT).    -   2. Vaccine or immunogenic composition for use in a method of        treating or preventing, in particular preventing Zika virus        infection in a human subject population in need thereof,        comprising administering to individual human subjects of said        human subject population the vaccine or immunogenic composition        comprising antigen from a Zika virus, wherein the vaccine or        immunogenic composition is administered as single dose or prime        administration and wherein the administration of the vaccine or        immunogenic composition induces 14 and/or 28 days after the        single dose or prime administration a seroconversion rate of at        least 25%, at least 30%, at least 40%, at least 50%, at least        60%, at least 70%, at least 75%, at least 80%, at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, or at least        90% in a population of at least 20 Zika virus seronegative human        subjects, as determined by the plaque reduction neutralization        test (PRNT).    -   3. Vaccine or immunogenic composition for use in a method of        treating or preventing, in particular preventing Zika virus        infection in a human subject population in need thereof,        comprising administering to individual human subjects of said        human subject population the vaccine or immunogenic composition        comprising antigen from a Zika virus, wherein the vaccine or        immunogenic composition is administered as single dose or prime        administration and wherein the administration of the vaccine or        immunogenic composition induces 14 and/or 28 days after the        single dose or prime administration a seropositivity rate of at        least 25%, at least 30%, at least 40%, at least 50%, at least        60%, at least 70%, at least 75%, at least 80%, at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, or at least        90% in a population of at least 20 Zika virus seronegative human        subjects or in a population of at least 20 Flavivirus naïve        human subjects, as determined by the plaque reduction        neutralization test (PRNT).    -   4. Vaccine or immunogenic composition for use in a method of        treating or preventing, in particular preventing Zika virus        infection in a human subject population in need thereof,        comprising administering to individual human subjects of said        human subject population the vaccine or immunogenic composition        comprising antigen from a Zika virus, wherein the vaccine or        immunogenic composition is administered as a single dose        administration or multi dose administration including at least a        first (prime) and a second (boost) administration and wherein        the administration of the vaccine or immunogenic composition        until 7 days after the administration induces systemic side        effects in less than 50% of a human subject population of at        least 20 flavivirus naïve human subjects or in a population of        at least 20 Zika virus seronegative human subjects.    -   5. Vaccine or immunogenic composition for use in a method of        treating or preventing, in particular preventing Zika virus        infection in a human subject population in need thereof,        comprising administering to individual human subjects of said        human subject population the vaccine or immunogenic composition        comprising antigen from a Zika virus obtainable by a method for        inactivating a Zika virus preparation comprising:        -   (a) isolating the Zika virus preparation from one or more            cells cultured in vitro, wherein the cells are used to            produce the Zika virus preparation, wherein isolating the            Zika virus preparation comprises one or more steps selected            from: (i) depth filtration, (ii) buffer exchange and/or            dilution; (iii) ion exchange chromatography; and        -   (b) treating the Zika virus preparation with formaldehyde,            optionally with formaldehyde, wherein the numerical result            of the multiplication of the formaldehyde concentration as            measured in % (w/v) with the period of incubation with            formaldehyde as measured in days is 0.025 to 0.5.    -   6. The vaccine or immunogenic composition of any one of items 1        to 5, wherein the vaccine or immunogenic composition is        administered as a first (prime) and a second (boost)        administration about 1 to about 16 weeks apart, and wherein the        administration of the vaccine or immunogenic composition induces        14 and/or 28 days after the boost administration geometric mean        neutralizing antibody titers in a population of at least 20        flavivirus naïve human subjects and/or in a population of at        least 20 Zika virus seronegative human subjects of greater than        300, or greater than 500, or greater than 1000, or greater than        1500, or greater than 2000, or greater than 3000, as determined        by the plaque reduction neutralization test (PRNT).    -   7. The vaccine or immunogenic composition of any one of items 1        to 6, wherein the vaccine or immunogenic composition is        administered as a first (prime) and a second (boost)        administration about 1 to about 16 weeks apart and wherein the        administration of the vaccine or immunogenic composition induces        14 and/or 28 days after the boost administration        -   a seroconversion rate of at least 70%, at least 75%, at            least 80%, at least 85%, at least 86%, at least 87%, at            least 88%, at least 89%, or at least 90%, at least 91%, at            least 92%, at least 93%, at least 94%, at least 95%, at            least 96%, at least 97%, at least 98%, at least 99% or 100%            in a population of at least 20 Zika virus seronegative human            subjects, as determined by the plaque reduction            neutralization test (PRNT),        -   and/or        -   a seropositivity rate of at least 70%, at least 75%, at            least 80%, at least 85%, at least 86%, at least 87%, at            least 88%, at least 89%, or at least 90%, at least 91%, at            least 92%, at least 93%, at least 94%, at least 95%, at            least 96%, at least 97%, at least 98%, at least 99% or 100%            in a population of at least 20 Zika virus seronegative human            subjects or in a population of at least 20 Flavivirus naïve            human subjects, as determined by the plaque reduction            neutralization test (PRNT).    -   8. The vaccine or immunogenic composition of any one of items 1        to 7, wherein the administration of the vaccine or immunogenic        composition until 7 days after the administration induces        headache symptoms in less than 29% of a human subject population        of at least 20 flavivirus naïve human subjects or of at least 20        Zika virus seronegative human subjects.    -   9. The vaccine or immunogenic composition of any one of items 1        to 8, wherein the administration of the vaccine or immunogenic        composition until 7 days after the administration induces fever        in 4% or less, and/or fatigue in 33% or less, and/or arthralgia        in 10% or less, and/or myalgia in 17% or less, and/or malaise in        15% or less of a human subject population of at least 20        flavivirus naïve human subjects or of at least 20 Zika virus        seronegative human subjects.    -   10. The vaccine or immunogenic composition of any one of items 1        to 9, wherein the vaccine or immunogenic composition is        administered as multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces systemic side effects in less than 40% of        a human subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   11. The vaccine or immunogenic composition of any one of items 1        to 10, wherein the vaccine or immunogenic composition is        administered as multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces        -   at least 40% less, or at least 45% less fatigue, and/or        -   no more fever, and/or        -   no more, or at least 10% less, or at least 20% less, or at            least 25% less myalgia, and/or        -   no more, or at least 10% less, or at least 20% less malaise    -    compared to 7 days after the prime administration in a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   12. The vaccine or immunogenic composition of any one of items 1        to 11, wherein the vaccine or immunogenic composition is        administered as a multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces fever in less than 4% or in 0% of a human        subject population of at least 20 flavivirus naïve human        subjects or at least 20 Zika virus seronegative human subjects.    -   13. The vaccine or immunogenic composition of any one of items 1        to 12, wherein the vaccine or immunogenic composition is        administered as multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces fatigue in less than 19% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   14. The vaccine or immunogenic composition of any one of items 1        to 13, wherein the vaccine or immunogenic composition is        administered as multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces myalgia in less than 12% or less than 8%        of a human subject population of at least 20 flavivirus naïve        human subjects or of at least 20 Zika virus seronegative human        subjects.    -   15. The vaccine or immunogenic composition of any one of items 1        to 14, wherein the vaccine or immunogenic composition is        administered as multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces malaise in less than 13% or in 10% or        less of a human subject population of at least 20 flavivirus        naïve human subjects or of at least 20 Zika virus seronegative        human subjects.    -   16. The vaccine or immunogenic composition of any one of items 1        to 15, wherein the vaccine or immunogenic composition is        administered as a multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces headache symptoms in 20% or less, and        arthralgia in 8% or less, and fever in less than 4%, and fatigue        in less than 19%, and myalgia in less than 12%, and malaise in        less than 13% of a human subject population of at least 20        flavivirus naïve human subjects or of at least 20 Zika virus        seronegative human subjects.    -   17. The vaccine or immunogenic composition of any one of claims        1 to 16, wherein the vaccine or immunogenic composition        comprises a dose of 1 μg to 40 μg of the antigen, wherein the        antigen is an inactivated whole virus.    -   18. The vaccine or immunogenic composition of claim 17, the Zika        virus having a mutation at position 98 of SEQ ID NO: 1, or at a        position corresponding to position 98 of SEQ ID NO: 1.    -   19. The vaccine or immunogenic composition of any one of claims        1 to 18, wherein the antigen is purified, and wherein the main        peak of the purified antigen in the size exclusion        chromatography is more than 85% of the total area under the        curve in the size exclusion chromatography.    -   20. The vaccine or immunogenic composition of any one of claims        1 to 19, wherein the vaccine or immunogenic composition is        administered to human subjects from a Zika endemic region,        optionally subject to an outbreak.    -   21. The vaccine or immunogenic composition of any one of claims        1 to 20, wherein the vaccine or immunogenic composition is        administered to human subjects from a Zika non-endemic region        travelling to an endemic region.    -   22. The vaccine or immunogenic composition of any one of claims        1 to 21, wherein the vaccine or immunogenic composition is        administered to human subjects 18 to 29 years of age, in        particular women of childbearing potential.    -   23. The vaccine or immunogenic composition of any one of claims        1 to 21, wherein the vaccine or immunogenic composition is        administered to human subjects 30 to 49 years of age, in        particular women of childbearing potential.    -   24. The vaccine or immunogenic composition of any one of items 1        to 23, wherein the vaccine or immunogenic composition is        administered as a single dose administration or multi dose        administration including at least a first (prime) and a second        (boost) administration wherein the vaccine or immunogenic        composition comprises a dose of about 5 μg of purified        inactivated whole virus.    -   25. The vaccine or immunogenic composition of claim 24 wherein        the administration of the vaccine or immunogenic composition        until 7 days after the single dose or prime administration        induces systemic side effects in less than 50%, or in less than        45%, or in less than 40% of a human subject population of at        least 20 flavivirus naïve human subjects or of at least 20 Zika        virus seronegative human subjects and/or wherein the        administration of the vaccine or immunogenic composition until 7        days after the boost administration induces systemic side        effects in less than 40%, or in less than 35%, or in less than        30%, or in less than 25%, or in less than 20%, or in less than        15% of a human subject population of at least 20 flavivirus        naïve human subjects or of at least 20 Zika virus seronegative        human subjects.    -   26. The vaccine or immunogenic composition of claim 24 or 25,        wherein the administration of the vaccine or immunogenic        composition until 7 days after the single dose or prime        administration induces fever in less than 3%, or 0%, of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces fever in less than 4%, or in less than        3%, or 0% of a human subject population of at least 20        flavivirus naïve human subjects or of at least 20 Zika virus        seronegative human subjects.    -   27. The vaccine or immunogenic composition of any one of items        24 to 26, herein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces headache in less than 29% of a        human subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces headache in less than 20%, or in less        than 15%, or in less than 10%, or in less than 5% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   28. The vaccine or immunogenic composition of any one of items        24 to 27, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces fatigue in less than 30%, or in        less than 25%, or in less than 20% of a human subject population        of at least 20 flavivirus naïve human subjects or of at least 20        Zika virus seronegative human subjects and/or wherein the        administration of the vaccine or immunogenic composition until 7        days after the boost administration induces fatigue in less than        20%, or in less than 15%, or in less than 10% of a human subject        population of at least 20 flavivirus naïve human subjects or of        at least 20 Zika virus seronegative human subjects.    -   29. The vaccine or immunogenic composition of any one of items        24 to 28, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces arthralgia in less than 4% of a        human subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces arthralgia in less than 5%, or in less        than 2%, or in 0% of a human subject population of at least 20        flavivirus naïve human subjects or of at least 20 Zika virus        seronegative human subjects.    -   30. The vaccine or immunogenic composition of any one of items        24 to 29, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces myalgia in 17% or less of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces myalgia in less than 12%, or in less than        10%, or in less than 5% of a human subject population of at        least 20 flavivirus naïve human subjects or of at least 20 Zika        virus seronegative human subjects.    -   31. The vaccine or immunogenic composition of any one of items        24 to 30, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces malaise in less than 10% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces malaise in less than 14%, or in less than        10%, or in less than 5%, or 0% of a human subject population of        at least 20 flavivirus naïve human subjects or of at least 20        Zika virus seronegative human subjects.    -   32. The vaccine or immunogenic composition of any one of items        24 to 31, wherein the vaccine or immunogenic composition is        administered as multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces        -   at least 70% less, or at least 60% less, or at least 50%            less, or at least 40% less, or at least 35% less, or at            least 30% less systemic side effects, and/or        -   no increase in fever, and/or        -   at least 80% less, or at least 70% less, or at least 60%            less, or at least 50% less, or at least 45% less headache,            and/or        -   at least 60% less, or at least 55% less, or at least 50%            less, or at least 45% less, or at least 40% less fatigue,            and/or        -   no increase in arthralgia, or at least 80% less, or at least            60% less, or at least 40% less, or at least 20% less, or at            least 10% less arthralgia, and/or        -   no increase in myalgia, or at least 70% less, or at least            60% less, or at least 40% less, or at least 20% less, or at            least 10% less myalgia, and/or        -   no increase in malaise, or at least 80% less, or at least            60% less, or at least 40% less, or at least 20% less, or at            least 10% less malaise        -   compared to 7 days after the prime administration in a human            subject population of at least 20 flavivirus naïve human            subjects or of at least 20 Zika virus seronegative human            subjects.    -   33. The vaccine or immunogenic composition of any one of items 1        to 23, wherein the vaccine or immunogenic composition is        administered as a single dose administration or multi dose        administration including at least a first (prime) and a second        (boost) administration wherein the vaccine or immunogenic        composition comprises a dose of about 10 μg of purified        inactivated whole virus.    -   34. The vaccine or immunogenic composition of claim 33, wherein        the administration of the vaccine or immunogenic composition        until 7 days after the single dose or prime administration        induces systemic side effects in less than 50% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces systemic side effects in less than 40%,        or in less than 35%, or in less than 30% of a human subject        population of at least 20 flavivirus naïve human subjects or of        at least 20 Zika virus seronegative human subjects.    -   35. The vaccine or immunogenic composition of claim 33 or 34,        wherein the administration of the vaccine or immunogenic        composition until 7 days after the single dose or prime        administration induces fever in less than 4% of a human subject        population of at least 20 flavivirus naïve human subjects or of        at least 20 Zika virus seronegative human subjects and/or        wherein the administration of the vaccine or immunogenic        composition until 7 days after the boost administration induces        fever in less than 4%, or in less than 3%, or 0% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   36. The vaccine or immunogenic composition of any one of items        33 to 35, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces headache in less than 29%, or in        less than 25%, or in less than 20%, or in less than 15% of a        human subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces headache in 20% or less of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   37. The vaccine or immunogenic composition of any one of items        33 to 36, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces fatigue in 33% or less of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces fatigue in less than 20% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   38. The vaccine or immunogenic composition of any one of items        33 to 37, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces arthralgia in 10% or less of a        human subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces arthralgia in less than 5% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   39. The vaccine or immunogenic composition of any one of items        33 to 38, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces myalgia in 17% or less of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces myalgia in less than 12%, or in less than        10% of a human subject population of at least 20 flavivirus        naïve human subjects or of at least 20 Zika virus seronegative        human subjects.    -   40. The vaccine or immunogenic composition of any one of items        33 to 39, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces malaise in 15% or less of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces malaise in less than 13%, or in 10% or        less of a human subject population of at least 20 flavivirus        naïve human subjects or of at least 20 Zika virus seronegative        human subjects.    -   41. The vaccine or immunogenic composition of any one of items        33 to 40, wherein the vaccine or immunogenic composition is        administered as multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces        -   at least 40% less, or at least 35% less, or at least 30%            less, or at least 25% less systemic side effects, and/or        -   no increase in fever, or at least 80% less, or at least 60%            less, or at least 40% less, or at least 20% less, or at            least 10% less fever, and/or        -   at least 45% less, or at least 40% less fatigue, and/or        -   no increase in arthralgia, or at least 65% less, or at least            60% less, or at least 40% less, or at least 20% less, or at            least 10% less arthralgia, and/or        -   no increase in myalgia, or at least 45% less, or at least            40% less, or at least 20% less, or at least 10% less            myalgia, and/or        -   no increase in malaise, or at least 20% less, or at least            10% less malaise        -   compared to 7 days after the prime administration in a human            subject population of at least 20 flavivirus naïve human            subjects or of at least 20 Zika virus seronegative human            subjects.    -   42. The vaccine or immunogenic composition of any one of items 1        to 23, wherein the vaccine or immunogenic composition is        administered as a single dose administration or multi dose        administration including at least a first (prime) and a second        (boost) administration wherein the vaccine or immunogenic        composition comprises a dose of about 2 μg of purified        inactivated whole virus.    -   43. The vaccine or immunogenic composition of claim 42, wherein        the administration of the vaccine or immunogenic composition        until 7 days after the single dose or prime administration        induces systemic side effects in less than 50%, or in less than        45%, or in less than 40%, or in less than 35% of a human subject        population of at least 20 flavivirus naïve human subjects or of        at least 20 Zika virus seronegative human subjects and/or        wherein the administration of the vaccine or immunogenic        composition until 7 days after the boost administration induces        systemic side effects in less than 40%, or in less than 35% of a        human subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   44. The vaccine or immunogenic composition of claim 42 or 43,        wherein the administration of the vaccine or immunogenic        composition until 7 days after the single dose or prime        administration induces fever in less than 3%, or 0%, of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces fever in less than 4%, or in less than        3%, or 0% of a human subject population of at least 20        flavivirus naïve human subjects or of at least 20 Zika virus        seronegative human subjects.    -   45. The vaccine or immunogenic composition of any one of items        42 to 44, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces headache in less than 29%, or in        less than 25%, or in less than 20% of a human subject population        of at least 20 flavivirus naïve human subjects or of at least 20        Zika virus seronegative human subjects and/or wherein the        administration of the vaccine or immunogenic composition until 7        days after the boost administration induces headache in less        than 20%, or in less than 15% of a human subject population of        at least 20 flavivirus naïve human subjects or of at least 20        Zika virus seronegative human subjects.    -   46. The vaccine or immunogenic composition of any one of items        42 to 45, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces fatigue in less than 30%, or in        less than 25% of a human subject population of at least 20        flavivirus naïve human subjects or of at least 20 Zika virus        seronegative human subjects and/or wherein the administration of        the vaccine or immunogenic composition until 7 days after the        boost administration induces fatigue in less than 20%, or in        less than 15% of a human subject population of at least 20        flavivirus naïve human subjects or of at least 20 Zika virus        seronegative human subjects.    -   47. The vaccine or immunogenic composition of any one of items        42 to 46, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces arthralgia in less than 4% of a        human subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces arthralgia in less than 8% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects.    -   48. The vaccine or immunogenic composition of any one of items        42 to 47, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces myalgia in 17% or less of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces myalgia in less than 12%, or in less than        10% of a human subject population of at least 20 flavivirus        naïve human subjects or of at least 20 Zika virus seronegative        human subjects.    -   49. The vaccine or immunogenic composition of any one of items        42 to 48, wherein the administration of the vaccine or        immunogenic composition until 7 days after the single dose or        prime administration induces malaise in less than 10% of a human        subject population of at least 20 flavivirus naïve human        subjects or of at least 20 Zika virus seronegative human        subjects and/or wherein the administration of the vaccine or        immunogenic composition until 7 days after the boost        administration induces malaise in less than 13%, or in less than        10%, or in less than 5%, or 0% of a human subject population of        at least 20 flavivirus naïve human subjects or of at least 20        Zika virus seronegative human subjects.    -   50. The vaccine or immunogenic composition of any one of items        42 to 49, wherein the vaccine or immunogenic composition is        administered as multiple doses in a first (prime) and a second        (boost) administration and wherein the administration of the        vaccine or immunogenic composition until 7 days after the boost        administration induces        -   no increase in fever, and/or        -   at least 50% less, or at least 45% less, or at least 40%            less fatigue, and/or        -   no increase in myalgia, or at least 20% less, or at least            10% less myalgia, and/or        -   no increase in malaise, or at least 80% less, or at least            60% less, or at least 40% less, or at least 20% less, or at            least 10% less malaise        -   compared to 7 days after the prime administration in a human            subject population of at least 20 flavivirus naïve human            subjects or of at least 20 Zika virus seronegative human            subjects.    -   51. The vaccine or immunogenic composition of any one of items 5        to 50, wherein the cells are non-human cells.    -   52. The vaccine or immunogenic composition of claim 51, wherein        the cells are Vero cells.    -   53. The vaccine or immunogenic composition of any one of items 5        to 52, wherein the Zika virus preparation is treated with        formaldehyde at a concentration of 0.005% (w/v) to 0.02% (w/v).    -   54. The vaccine or immunogenic composition of any one of items 5        to 53, wherein the Zika virus preparation is treated for eight        to twelve days.    -   55. The vaccine or immunogenic composition of claim 54, wherein        the Zika virus preparation is treated for ten days.    -   56. The vaccine or immunogenic composition of any one of items 5        to 55, wherein the Zika virus preparation is treated at a        temperature of 15° C. to 30° C.    -   57. The vaccine or immunogenic composition of claim 56, wherein        the Zika virus preparation is treated at a temperature of 22° C.    -   58. The vaccine or immunogenic composition of any one of items 5        to 57, further comprising a step (c) of determining the        completeness of inactivation.    -   59. The vaccine or immunogenic composition of claim 58, wherein        step (c) comprises:        -   (i) inoculating cultured insect cells with a Zika virus            preparation treated according to step (b) and incubating the            insect cells for a first period of time, thereby producing            an insect cell supernatant;        -   (ii) inoculating cultured mammalian cells with the insect            cell supernatant produced in (i) and incubating the            mammalian cells for a second period of time; and        -   (iii) determining whether the Zika virus preparation            contains a residual replicating virus that produces a            cytopathic effect on the mammalian cells.    -   60. The vaccine or immunogenic composition of claim 59, wherein        the insect cells are selected from CCL-125 cells, Aag-2 cells,        RML-12 cells, C6/36 cells, C7-10 cells, AP-61 cells, A.t. GRIP-1        cells, A.t. GRIP-2 cells, A.t. GRIP-3 cells, UM-AVE1 cells,        Mos.55 cells, Sua1B cells, 4a-3B cells, Mos.42 cells, MSQ43        cells, LSB-AA695BB cells, NIID-CTR cells and TRA-171 cells, such        as C6/36 cells.    -   61. The vaccine or immunogenic composition of claim 59 or 60,        wherein the first period of time is 3 to 7 days.    -   62. The vaccine or immunogenic composition of any one of items        59 to 61, wherein the mammalian cells are selected from VERO        cells, LLC-MK2 cells, MDBK cells, MDCK cells, ATCC CCL34 MDCK        (NBL2) cells, MDCK 33016 (deposit number DSM ACC 2219 as        described in WO97/37001) cells, BHK21-F cells, HKCC cells, and        Chinese hamster ovary cells (CHO cells), such as VERO cells.    -   63. The vaccine or immunogenic composition of any one of items        59 to 62, wherein the second period of time is 3 to 14 days.    -   64. The vaccine or immunogenic composition of any one of items 5        to 63, further comprising a step (d) of neutralizing the        formaldehyde-treated Zika virus preparation with sodium        metabisulfite.    -   65. The vaccine or immunogenic composition of claim 64, wherein        the formaldehyde-treated Zika virus preparation is neutralized        at least five, at least seven, at least nine, at least 11, or at        least 14 days after formaldehyde treatment.    -   66. The vaccine or immunogenic composition of any one of items 1        to 65, wherein the vaccine or immunogenic composition has a        residual formaldehyde content of less than 50 μg/ml.

1-68. (canceled)
 69. A method of treating or preventing Zika virusinfection in a human subject population in need thereof, comprisingadministering to individual human subjects of said human subjectpopulation a vaccine or immunogenic composition comprising antigen froma Zika virus, wherein the vaccine or immunogenic composition isadministered as a single dose or prime administration, and wherein theadministration of the vaccine or immunogenic composition induces 14and/or 28 days after the single dose or prime administration geometricmean neutralizing antibody titers in a population of at least 20flavivirus naïve human subjects and/or in a population of at least 20Zika virus seronegative human subjects of greater than 10, as determinedby the plaque reduction neutralization test (PRNT).
 70. A Method oftreating or preventing Zika virus infection in a human subjectpopulation in need thereof, comprising administering to individual humansubjects of said human subject population a vaccine or immunogeniccomposition comprising antigen from a Zika virus, wherein the vaccine orimmunogenic composition is administered as single dose or primeadministration and wherein the administration of the vaccine orimmunogenic composition induces 14 and/or 28 days after the single doseor prime administration a seroconversion rate of at least 25%, in apopulation of at least 20 Zika virus seronegative human subjects, asdetermined by the plaque reduction neutralization test (PRNT).
 71. Themethod of claim 70 wherein within 14 and/or 28 days after the singledose or prime administration a seropositivity rate of at least 25%, isinduced in a population of at least 20 Zika virus seronegative humansubjects or in a population of at least 20 Flavivirus naïve humansubjects, as determined by the plaque reduction neutralization test(PRNT).
 72. The Method of claim 70, wherein the vaccine or immunogeniccomposition is administered as a single dose administration or multidose administration including at least a first (prime) and a second(boost) administration and wherein the administration of the vaccine orimmunogenic composition until 7 days after the administration inducessystemic side effects in less than 50% of a human subject population ofat least 20 flavivirus naïve human subjects or in a population of atleast 20 Zika virus seronegative human subjects.
 73. The method of claim69, wherein the vaccine or immunogenic composition is administered as afirst (prime) and a second (boost) administration about 1 to about 16weeks apart, and wherein the administration of the vaccine orimmunogenic composition induces 14 and/or 28 days after the boostadministration geometric mean neutralizing antibody titers in apopulation of at least 20 flavivirus naïve human subjects and/or in apopulation of at least 20 Zika virus seronegative human subjects ofgreater than 300, as determined by the plaque reduction neutralizationtest (PRNT).
 74. The method of claim 70, wherein the vaccine orimmunogenic composition is administered as a first (prime) and a second(boost) administration about 1 to about 16 weeks apart and wherein theadministration of the vaccine or immunogenic composition induces 14and/or 28 days after the boost administration a seroconversion rate ofat least 70% in a population of at least 20 Zika virus seronegativehuman subjects, as determined by the plaque reduction neutralizationtest (PRNT), and a seropositivity rate of at least 70% in a populationof at least 20 Zika virus seronegative human subjects or in a populationof at least 20 Flavivirus naïve human subjects, as determined by theplaque reduction neutralization test (PRNT).
 75. The method of claim 70,wherein the administration of the vaccine or immunogenic compositionuntil 7 days after the administration induces headache symptoms in lessthan 29% of a human subject population of at least 20 flavivirus naïvehuman subjects or of at least 20 Zika virus seronegative human subjects.76. The method of claim 70, wherein the administration of the vaccine orimmunogenic composition until 7 days after the administration inducesfever in 4% or less, and/or fatigue in 33% or less, and/or arthralgia in10% or less, and/or myalgia in 17% or less, and/or malaise in 15% orless of a human subject population of at least 20 flavivirus naïve humansubjects or of at least 20 Zika virus seronegative human subjects. 77.The method of claim 70, wherein the vaccine or immunogenic compositionis administered as multiple doses in a first (prime) and a second(boost) administration and wherein the administration of the vaccine orimmunogenic composition until 7 days after the boost administrationinduces systemic side effects in less than 40% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects.
 78. The method of claim 70,wherein the vaccine or immunogenic composition is administered asmultiple doses in a first (prime) and a second (boost) administrationand wherein the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces at least 40% less,or at least 45% less fatigue, and/or no more fever, and/or no more, orat least 10% less, or at least 20% less, or at least 25% less myalgia,and/or no more, or at least 10% less, or at least 20% less malaisecompared to 7 days after the prime administration in a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects.
 79. The method of claim 70,wherein the vaccine or immunogenic composition is administered asmultiple doses in a first (prime) and a second (boost) administrationand wherein the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces fever in less than4% or in 0% of a human subject population of at least 20 flavivirusnaïve human subjects or at least 20 Zika virus seronegative humansubjects.
 80. The method of claim 70, wherein the vaccine or immunogeniccomposition is administered as multiple doses in a first (prime) and asecond (boost) administration and wherein the administration of thevaccine or immunogenic composition until 7 days after the boostadministration induces fatigue in less than 19% of a human subjectpopulation of at least 20 flavivirus naïve human subjects or of at least20 Zika virus seronegative human subjects.
 81. The method of claim 70,wherein the vaccine or immunogenic composition is administered asmultiple doses in a first (prime) and a second (boost) administrationand wherein the administration of the vaccine or immunogenic compositionuntil 7 days after the boost administration induces myalgia in less than12% or less than 8% of a human subject population of at least 20flavivirus naïve human subjects or of at least 20 Zika virusseronegative human subjects.
 82. The method of claim 70, wherein thevaccine or immunogenic composition is administered as multiple doses ina first (prime) and a second (boost) administration and wherein theadministration of the vaccine or immunogenic composition until 7 daysafter the boost administration induces malaise in less than 13% or in10% or less of a human subject population of at least 20 flavivirusnaïve human subjects or of at least 20 Zika virus seronegative humansubjects.
 83. The method of claim 70, wherein the vaccine or immunogeniccomposition is administered as multiple doses in a first (prime) and asecond (boost) administration and wherein the administration of thevaccine or immunogenic composition until 7 days after the boostadministration induces headache symptoms in 20% or less, and arthralgiain 8% or less, and fever in less than 4%, and fatigue in less than 19%,and myalgia in less than 12%, and malaise in less than 13% of a humansubject population of at least 20 flavivirus naïve human subjects or ofat least 20 Zika virus seronegative human subjects.
 84. The method ofclaim 70, wherein the vaccine or immunogenic composition comprises adose of 1 μg to 40 μg of the antigen, wherein the antigen is aninactivated whole virus.
 85. The method of claim 84, the Zika virushaving a mutation at position 98 of SEQ ID NO: 1, or at a positioncorresponding to position 98 of SEQ ID NO:
 1. 86. The method of claim70, wherein the antigen is purified, and wherein the main peak of thepurified antigen in the size exclusion chromatography is more than 85%of the total area under the curve in the size exclusion chromatography.87. The method of claim 70, wherein the vaccine or immunogeniccomposition is administered to human subjects from a Zika endemicregion, optionally subject to an outbreak.
 88. The method of claim 70,wherein the vaccine or immunogenic composition is administered to humansubjects from a Zika non-endemic region travelling to an endemic region.89. The method of claim 70, wherein the vaccine or immunogeniccomposition is administered to human subjects 18 to 29 years of age, inparticular women of childbearing potential.
 90. The method of claim 70,wherein the vaccine or immunogenic composition is administered to humansubjects 30 to 49 years of age, in particular women of childbearingpotential.